Methods and compositions for improving plant health

ABSTRACT

The present invention provides methods and compositions for improving plant health. In particular, application of dicamba or another substrate of DMO, or metabolites thereof including DCSA, to a plant confers tolerance to, or defense against, abiotic or biotic stresses such as oxidative stress including herbicide application, and plant disease, and enhances crop yield. Such application may be in combination with the application of another herbicide such as glyphosate.

This application is a continuation-in-part of International ApplicationNo. PCT/US2007/081527, filed Oct. 16, 2007, which claims the priority ofU.S. Provisional Application Ser. No. 60/852,308 filed Oct. 16, 2006,the entire disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of agriculture. Morespecifically, the invention relates to methods for improving planthealth by application of dicamba herbicide and/or metabolites or analogsthereof to plants.

2. Description of Related Art

Dicamba (3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoicacid; FIG. 7), active ingredient in herbicides such as Banvel® (BASF),Clarity® (BASF), and Vanquish® (Syngenta), is a potent herbicide.Although its precise mechanism of activity is unclear, it appears to actas a plant growth regulator (e.g. Grossmann 2000). Its applicationresults in uncontrolled growth, leaf curling and twisting, chloroplastdamage, and direct phytotoxic effects, among others. Some of theseeffects are believed to be caused by ethylene synthesis, which triggersan increase in the biosynthesis of abscisic acid, another plant hormone.Thus imbalances in plant hormone levels appear to underlie the toxiceffects.

A dicamba monooxygenase (DMO), has been found to confer tolerance todicamba by degrading it to 3,6-dichloro salicylic acid (DCSA; 3,6-DCSA;FIG. 7) in bacteria (e.g. Herman et al., 2005; US Patent Publ.20060168700; U.S. Pat. No. 7,022,896). The DMO gene has subsequentlybeen used to confer tolerance to Dicamba in soybean and other plants(e.g. Weeks et al., 2006). These new dicamba-tolerant crops allow forapplications of dicamba on crops which were previously extremelysensitive to any dicamba exposure, particularly dicots such as cotton,canola, and soybean.

U.S. Pat. No. 7,230,163 describes a method of improving crop yields byapplication of a synthetic auxin to a plant. However, plant health, e.g.resistance to biotic or abiotic stress, is not addressed, nor is theeffect of metabolites of the applied auxin.

As in planta metabolism is generally unpredictable, i.e., one cannotpredict from prior traditional uses of dicamba (e.g., on corn notexpressing a DMO gene) what metabolites might result from the use ofdicamba in new dicamba-tolerant crops expressing DMO, or the effects ofsuch metabolites in the plant. The present inventors have found that inthese DMO-expressing crops, DMO detoxifies dicamba and produces dicambametabolites, including DCSA. Unexpectedly, the present inventors havealso discovered that this DCSA metabolite confers beneficial healtheffects on the plants. Accordingly, the present invention is directedto, among other things, methods of improving plant health using dicambametabolites, including DCSA, and the plants, seeds, and crops resultingtherefrom.

These improvements in plant health provide increased resistance ofplants against biotic (e.g., insects, fungi, viruses, nematodes, andother pathogens) and abiotic stresses (e.g., drought, cold, ozone, soilnutrient deficiencies), with resulting increases in yields and improvedquality of crops, all of which will be a great benefit to agriculture.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for improving the healthof a plant, comprising providing the plant with dicamba, or a product ofDMO-mediated metabolism or analog thereof, in an amount that improvesthe health of the plant as compared to a plant of the same genotype notprovided with the dicamba or product of DMO-mediated metabolism oranalog thereof. In one embodiment, the invention provides a method forimproving the health of a plant, comprising contacting the plant withdicamba or a product of DMO-mediated metabolism thereof to provide theplant with a product of DMO-mediated metabolism of dicamba in an amountthat improves the health of the plant as compared to a plant of the samegenotype not provided with the dicamba or product of DMO-mediatedmetabolism thereof, wherein a plant contacted with dicamba comprisesDMO.

In one embodiment, the plant is in a crop production field. The methodmay further comprise allowing the plant to be subject to biotic orabiotic stress prior to, concurrently with or after providing the plantwith the dicamba or a product of DMO-mediated metabolism or analogthereof. The method may also further comprise the step of identifyingthe plant as in need of improved plant health prior to providing theplant with the dicamba or a product of DMO-mediated metabolism or analogthereof.

In one embodiment, identifying the plant as in need of improved planthealth comprises identifying the plant as comprising at least a firstsymptom indicative of biotic or abiotic stress. In specific embodiments,the symptom is selected from the group consisting of stunting, loss ofphotosynthetic function, lipid peroxidation, accumulation of activeoxygen species, increase in free radical content, and tissuenecrotization (also termed the “hypersensitive response”). In aparticular embodiment, the plant displays a symptom of stunting. Theplant identified as in need of improved health may be an immature plantundergoing vegetative growth and sensitive to a disease or to weedgrowth. In particular embodiments, the plant is a soybean plant ingrowth stage VE to V3, a cotton plant prior to 1^(st) square formation,or a corn plant prior to or during growth stages VE to VT.

The plant health of which is improved according to the invention may beat risk for or under abiotic stress. Examples of abiotic stress includeosmotic stress, heat or cold exposure, oxidative stress and nutrientdeficit. In a particular embodiment, the plant is at risk for or underosmotic stress, such as drought stress. In another embodiment, the plantis at risk for or under oxidative stress, such as due to application ofan herbicide, or due to the presence of ozone at a level that can injurea plant.

Such a plant may also be defined as at risk for or under biotic stress.Examples of biotic stress include fungal disease such as Soybean Rust,viral disease, bacterial disease, insect infestation, nematodeinfestation, and weed infestation. In one embodiment, the plant is atrisk for or under the stress of fungal disease. In a particularembodiment, the plant is at risk for or under the stress of SoybeanRust. In another embodiment, the plant is at risk for or under thestress of weed infestation. Weed infestation constitutes a stress to acrop, especially to young plants such as soybean plants up to growthstage V3-V4.

In a method of the invention, tolerance to oxidative stress in a plantmay be increased. A method of the invention may also comprise providinga population of plants with the dicamba or a product of DMO-mediatedmetabolism or analog thereof to improve the health of the plants, forinstance by metabolism of dicamba to DCSA. If dicamba is applied, thismay be at a rate which is not herbicidal, yet results in a plant healthbenefit due to metabolism of dicamba to DCSA, or other product of plantmetabolism of dicamba or DCSA.

In a method of the invention, the product of DMO-mediated metabolism maybe defined as one or more of 3,6-DCSA, 3,5-DCSA, or 3-CSA, or ametabolite of 3,6-DCSA, 3,5-DCSA, and 3-CSA. The dicamba, appliedproduct of DMO-mediated metabolism, or a metabolite, may be herbicidal.The plant may comprise a transgene that encodes DMO. The applied productor a metabolite of the applied product may also be non-herbicidal. Inone embodiment, an application rate to a field is used from about 0.0025pounds per Acre (lb/A) to about 9 lb/A, including about 0.25 lb/A toabout 1.5 lb/A of dicamba, including, for example, from about 0.25 lb/Ato about 1 lb/A, from about 0.5 lb/A to about 1.5 lb/A, and from about0.5 lb/A to about 1 lb/A, as well as lower or higher amounts and allranges therebetween, such as about 0.005 lb/A, about 0.01 lb/A, about0.025 lb/A, about 0.05 lb/A, about 0.15 lb/A, about 0.175 lb/A, about 2lb/A, about 4 lb/A, about 7 lb/A, and about 12 lb/A. In anotherembodiment, from about 0.0025 lb/A to about 12 lb/A of DCSA is used,including, for example, from about 0.25 lb/A to about 4 lb/A, from about0.5 lb/A to about 6 lb/A, and from about 4 lb/A to about 12 lb/A, aswell as lower or higher amounts and all ranges therebetween, includingabout 0.005 lb/A, about 0.01 lb/A, about 0.025 lb/A, about 0.05 lb/A,about 0.15 lb/A, about 0.175 lb/A, about 0.25 lb/A, about 0.5 lb/A,about 1 lb/A, about 3 lb/A, about 5 lb/A, about 8 lb/A, about 12 lb/A,about 15 lb/A, and about 20 lb/A. In certain embodiments of theinvention, the dicamba or the product of DMO-mediated metabolism may beapplied repeatedly, as well as at a non-herbicidal application rate.

In specific embodiments DCSA is provided to a plant, including any partthereof, by applying dicamba to the plant and allowing the metabolism ofthe plant to produce DCSA. In this way, health benefits can be obtainedby direct application of dicamba. Alternatively, DCSA or a differentproduct of the metabolism of dicamba may be administered directly.

A plant used in an embodiment of the invention may comprise a transgenethat encodes DMO. The plant may be a dicotyledonous plant. Examples ofdicotyledonous plants include alfalfa, beans, beet, broccoli, cabbage,canola, carrot, cauliflower, celery, Chinese cabbage, cotton, cucumber,eggplant, flax, Jerusalem artichoke, lettuce, lupine, melon, pea,pepper, peanut, potato, pumpkin, radish, rapeseed, spinach, soybean,squash, sugarbeet, sunflower, tomato, and watermelon. In certainembodiments, the plant is selected from the group consisting of cotton,canola, or a legume such as soybean or alfalfa. In a particularembodiment, the plant is a soybean plant (Glycine max). In anotherembodiment, the plant is a cotton plant (Gossypium sp., such as G.hirsutum).

In certain embodiments the plant may be a monocotyledonous plant.Examples of monocotyledonous plants include barley, corn, leek, onion,rice, sorghum, sweet corn, wheat, rye, millet, sugarcane, oat,triticale, switchgrass, and turfgrass. In certain embodiments, the plantmay be a cereal (Gramineae), such as corn (Zea mays). In a particularembodiment, the plant is corn.

The plant may further be defined as tolerant to a herbicide selectedfrom the group consisting of glyphosate, glufosinate, 2,4-D,isoxaflutole, dicamba, and sulfonylurea, including any combinationsthereof, and may be treated with any such herbicides. In certainembodiments, the plant is tolerant to glyphosate and sulfonylurea, or toglufosinate and sulfonylurea. In yet other embodiments, the plant istolerant to glyphosate and dicamba. In a particular embodiment, theplant is tolerant to dicamba through the presence of a transgene thatdetoxifies dicamba. In another particular embodiment, the plant does notcomprise a transgene that detoxifies dicamba.

In yet another aspect, the invention provides a method for enhancing theyield of a plant comprising contacting the plant with an amount ofdicamba, or a product of DMO-mediated metabolism of dicamba, effectiveto increase the yield of the plant relative to a plant of the samegenotype grown under the same conditions but not contacted with thedicamba or a product of DMO-mediated metabolism of dicamba. The plantmay be in a crop production field. The method may further comprisecontacting a population of plants with the dicamba or a product ofDMO-mediated metabolism thereof. The product of DMO-mediated metabolismmay be 3,6-DCSA, 3,5-DCSA, or 3-CSA, or a metabolite of 3,6-DCSA,3,5-DCSA, or 3-CSA, such as DCGA (5-OH DCSA; DC-gentisic acid).

In specific embodiments of the method, the plant may contain a transgenethat encodes DMO. The plant may be, for example, a dicotyledonous ormonocotyledonous plant as set forth herein. The plant may further bedefined as tolerant to a herbicide selected from the group consisting ofglyphosate, glufosinate, 2,4-D, mesotrione, thiazopyr, isoxaflutole,bromoxynil, atrazine, fluazifop-P, and sulfonylureas/imidazolinones,including any combination thereof, and may be treated with any suchherbicides.

Another embodiment of the invention comprises a method for improving thehealth of a seed, comprising contacting the seed with dicamba or aproduct of DMO-mediated metabolism thereof in an amount that improvesthe health of the seed as compared to a seed of the same genotype notcontacted with the dicamba or product of DMO-mediated metabolismthereof.

In still yet another aspect, the invention provides a method ofproducing 3,6-DCSA comprising contacting a population of plants in acrop production field with dicamba, wherein the plants comprise atransgene encoding DMO.

In yet another aspect, the invention provides a method for improving thehealth of a plant exposed to a heavy metal, comprising contacting theplant with DCSA in combination with the heavy metal or prior to theheavy metal application or with dicamba or a product of DMO-mediatedmetabolism thereof in an amount that improves the health of the heavymetal treated plant as compared to a plant of the same genotype notcontacted with DCSA, dicamba or product of DMO-mediated metabolismthereof.

Another embodiment of the invention comprises a method for improving thehealth of a plant treated with or exposed to AMPA, comprising contactingthe plant with DCSA in combination with glyphosate or prior to theglyphosate treatment to a glyphosate tolerant plant or application ofdicamba or a product of DMO-mediated metabolism thereof in an amountthat improves the health of the glyphosate treated glyphosate tolerantplant as compared to a plant of the same genotype not contacted withDCSA, dicamba or product of DMO-mediated metabolism thereof.

In still yet another aspect, the invention provides a method forincreasing the germination rate of a seed, comprising contacting theseed with dicamba or a product of DMO-mediated metabolism thereof in anamount that improves the germination of the seed as compared to a seedof the same genotype not contacted with the dicamba or a product ofDMO-mediated metabolism thereof. In one embodiment, the seed may be in acrop production field. In another embodiment, the method may furthercomprise contacting a population of seeds with the dicamba or a productof DMO-mediated metabolism thereof. In a particular embodiment, the seedmay be coated with a composition comprising the dicamba or a product ofDMO-mediated metabolism thereof. The product of DMO-mediated metabolism,in one embodiment of the invention, may be one or more of 3,6-DCSA,3,5-DCSA, or 3-CSA, or a metabolite of 3,6-DCSA, 3,5-DCSA, or 3-CSA. Inanother embodiment, the product may be an analog of DCSA. In a certainembodiment, the dicamba or a product of DMO-mediated metabolism dicambamay be herbicidal and the plant may comprise a transgene that encodesDMO. The dicamba or a product of DMO-mediated metabolism dicamba may, inanother embodiment, may be non-herbicidal.

In one embodiment, the seed is treated with about 0.1 grams to about 100grams of dicamba or a product of DMO-mediated metabolism, including forexample, from about 0.1 grams to about 95 grams, about 0.1 grams toabout 50 grams, about 0.1 grams to about 105 grams, about 0.1 grams toabout 150 grams, about 0.05 grams to about 100 grams, about 0.5 grams toabout 100 grams, as well as lower or higher amounts and all rangestherebetween, including about 0.001 grams, about 0.005 grams, about 0.01grams, about 0.025 grams, about 0.05 grams, about 0.075 grams, about0.125 grams, about 0.15 grams, about 0.175 grams, about 1.0 grams, about3.0 grams, about 5.0 grams, about 10 grams, about 25 grams, about 75grams, about 90 grams, about 99, grams, about 101 grams about 110 grams,about 125 grams, per about 100 kilograms of seed, or amounts thereabout,for example, about 50 kilograms, about 75 kilograms, about 80 kilograms,about 0 kilograms, about 95 kilograms, about 96 kilograms, about 97kilograms, about 98 kilograms, about 99 kilograms, about 101 kilograms,about 102 kilograms, about 103 kilograms, about 104 kilograms, about 105kilograms, about 110 kilograms, about 120 kilograms, about 125kilograms, and about 150 kilograms.

In a specific embodiment, the seed may be from a dicotyledonous plant,for example alfalfa, beans, beet, broccoli, cabbage, carrot,cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant, flax,lettuce, lupine, melon, pea, pepper, peanut, potato, pumpkin, radish,rapeseed, spinach, soybean, squash, sugarbeet, sunflower, tomato, andwatermelon. In another embodiment, the seed may be from amonocotyledonous plant, for example, barley, corn, leek, onion, rice,sorghum, sweet corn, wheat, rye, millet, sugarcane, oat, triticale,switchgrass, and turfgrass. In one embodiment, the seed may be a seed ofa plant tolerant to a herbicide selected from the group consisting ofglyphosate, glufosinate, 2,4-D, mesotrione, dithiopyr, isoxaflutole,bromoxynil, atrazine, fluazifop-P, and sulfonylureas/imidazolinones. Inyet another embodiment, the seed may be contacted with at least oneherbicide, for example glyphosate, glufosinate, 2,4-D, mesotrione,thiazopyr, isoxaflutole, bromoxynil, atrazine, fluazifop-P, andsulfonylureas/imidazolinones.

In still yet another aspect, the invention provides a method forreducing or preventing yellow flash in a plant, comprising contactingthe plant with dicamba or a product of DMO-mediated metabolism thereofin an amount that reduces or prevents the yellow flash. In oneembodiment, the invention further comprises identifying the plant asexhibiting yellow flash or at risk of exhibiting yellow flash prior tocontacting the plant with dicamba or a product of DMO-mediatedmetabolism. The plant may be in a crop production field. In anotherembodiment, a population of plants may be contacted with the dicamba ora product of DMO-mediated metabolism thereof. In specific embodiments,the product of DMO-mediated metabolism is 3,6-DCSA, 3,5-DCSA, or 3-CSA,or a metabolite of 3,6-DCSA, 3,5-DCSA, or 3-CSA, and in anotherembodiment, it is an analog of DCSA. The product of DMO-mediatedmetabolism of dicamba may be herbicidal and the plant may comprise atransgene that encodes DMO, in another embodiment, the product is notherbicidal.

In yet another embodiment, the plant comprises a transgene conferringglyphosate tolerance. The plant may, in one embodiment, be contactedwith a tank mix comprising glyphosate and the dicamba or a product ofDMO-mediated metabolism thereof and the glyphosate may be present in anamount that would damage the plant in the absence of the dicamba or aproduct of DMO-mediated metabolism thereof.

In one embodiment the plant may a dicotyledonous plant, such as alfalfa,beans, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinesecabbage, cotton, cucumber, eggplant, flax, lettuce, lupine, melon, pea,pepper, peanut, potato, pumpkin, radish, rapeseed, spinach, soybean,squash, sugarbeet, sunflower, tomato, and watermelon. In anotherembodiment, the plant is a monocotyledonous plant, such as barley, corn,leek, onion, rice, sorghum, sweet corn, wheat, rye, millet, sugarcane,oat, triticale, switchgrass, and turfgrass. The plant, in oneembodiment, may be tolerant to a herbicide, such as glyphosate,glufosinate, 2,4-D, mesotrione, dithiopyr, isoxaflutole, bromoxynil,atrazine, fluazifop-P, and sulfonylureas/imidazolinones and in anotherembodiment may be contacted with at least one herbicide, for exampleglyphosate, glufosinate, 2,4-D, mesotrione, thiazopyr, isoxaflutole,bromoxynil, atrazine, fluazifop-P, and sulfonylureas/imidazolinones.

In still yet another aspect, the invention provides a method forreducing or preventing the deleterious health effect of a heavy metal orheavy metal salt on a plant, comprising contacting the plant withdicamba or a product of DMO-mediated metabolism thereof in an amountthat reduces or prevents the deleterious health effect of exposure ofthe plant to the heavy metal or heavy metal salt. In one embodiment, thedicamba or a product of DMO-mediated metabolism thereof to the plant ina composition that comprises the heavy metal or heavy metal salt. In afurther embodiment, the composition comprises an active including, forexample, a fungicide, a herbicide, a nematicide and an insecticide.

In one embodiment, the invention further comprises the step ofidentifying the plant as being exposed to the heavy metal or heavy metalsalt or at risk for exposure to the heavy metal or heavy metal salt. Theplant may be in a crop production field. In a certain embodiment, theheavy metal may be selected from the group consisting of copper, iron,aluminum, lead, mercury, cadmium, manganese, nickel, and zinc. Inspecific embodiments, the product of DMO-mediated metabolism is3,6-DCSA, 3,5-DCSA, or 3-CSA, or a metabolite of 3,6-DCSA, 3,5-DCSA, or3-CSA, the product may also be an analog of DCSA. The product mayfurther be herbicidal and the plant may comprise a transgene thatencodes DMO, or the product may not be herbicidal.

A plant used in one embodiment may be a dicotyledonous plant. Examplesof dicotyledonous plants include, for example, alfalfa, beans, beet,broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cotton,cucumber, eggplant, flax, lettuce, lupine, melon, pea, pepper, peanut,potato, pumpkin, radish, rapeseed, spinach, soybean, squash, sugarbeet,sunflower, tomato, and watermelon. In another embodiment, the plant maybe a monocotyledonous plant. Examples of monocotyledonous plantsinclude, for example, barley, corn, leek, onion, rice, sorghum, sweetcorn, wheat, rye, millet, sugarcane, oat, triticale, switchgrass, andturfgrass.

In another embodiment the plant may be tolerant to a herbicide, forexample glyphosate, glufosinate, 2,4-D, mesotrione, dithiopyr,isoxaflutole, bromoxynil, atrazine, fluazifop-P, andsulfonylureas/imidazolinones and may be contacted with at least oneherbicide, for example glyphosate, glufosinate, 2,4-D, mesotrione,thiazopyr, isoxaflutole, bromoxynil, atrazine, fluazifop-P, andsulfonylureas/imidazolinones.

In still yet another aspect, the invention provides a method forproducing doubled haploid plant tissue comprising the steps of a)obtaining a haploid plant tissue; b) treating the haploid plant tissuewith a chromosome doubling agent; and c) contacting the haploid planttissue or a doubled haploid tissue obtained therefrom prior to,concurrently with or subsequent to step b) with a composition comprisingdicamba, a product of DMO-mediated metabolism thereof, acetyl salicylicacid, salicylic acid or combinations thereof, in an amount thatincreases the efficiency with which doubled haploid tissue is obtainedrelative to a haploid tissue treated with the same conditions withoutsaid composition. In specific embodiments, the chromosome doubling agentmay be for instance nitrous oxide gas, an anti-microtubule herbicide, ananti-microtubule agent, and a mitotic inhibitor or may further beamiprophosmethyl (APM), pronamide, oryzalin, trifluralin, colchicine,griseofulvin, taxanes, paclitaxel, docetaxel, vinca alkaloids,vinblastine, vincristine, and vinorelbine. In another embodiment, theproduct of DMO-mediated metabolism may be 3,6-DCSA, 3,5-DCSA, or 3-CSA,or a metabolite of 3,6-DCSA, 3,5-DCSA, or 3-CSA.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 induction of PR2 at various time points after treating DMO plantswith dicamba.

FIG. 2 induction of PR2 at various rates of dicamba and DCSA 24 hrsafter treatment in two independent transgenic lines of soybean.

FIG. 3 effect of treating cotton plants with Pythium ultimum, in thepresence or absence of 3,6-DCSA.

FIG. 4 effect of treating cotton plants with Xanthomonas campestris pv.malvacearum in the presence or absence of 3,6-DCSA.

FIG. 5 lack of adverse effect on yield and other agronomic traits ofproviding a DMO gene in soybean.

FIG. 6 dicamba spray effect on yield.

FIG. 7 chemical structures of dicamba and 3,6-DCSA.

FIG. 8. soybean yield/dicamba efficacy trials average of 15 locations.

FIG. 9. results of 2,4-D postemergence treatment at V3 on transgenic andnon-transgenic soybean plants.

FIG. 10. metabolism of ¹⁴C-dicamba to DCSA in soy leaf strips in 24hours.

FIG. 11. metabolism of ¹⁴C-dicamba to DCSA and conjugation to glucosidein whole plant studies.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, methods are provided for improvingplant health, including increasing disease resistance of a plant,conferring enhanced tolerance to oxidative stress on a plant, and/orenhancing the yield of a plant. The plant health and other benefits ofproviding dicamba, or a metabolite of dicamba such as 3,6-DCSA, or DCGA(5-OH DCSA; DC-gentisic acid) are particularly surprising given thatdicamba is normally highly toxic to many plant species. “Dicamba” refersto 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxy benzoic acid(FIG. 7) and its acids and salts. Its salts include isopropylamine,diglycoamine, dimethylamine, potassium and sodium. Examples ofcommercial formulations of dicamba include, without limitation, Banvel™(as DMA salt), Clarity® (as DGA salt, BASF), VEL-58-CS-11™ and Vanquish™(as DGA salt, BASF).

The invention therefore relates, in one aspect, to the surprisingdiscovery that treatment with dicamba to a dicamba resistant plant,comprising a dicamba monooxygenase (DMO) transgene, confers improvedhealth to the plant. Such health benefits may include, for example,resistance to biotic and abiotic stresses to the plant. In specificembodiments, dicamba, 3,6-DCSA (see, e.g., FIG. 7), and analogs thereofmay be used to obtain one or more health benefit selected from diseaseresistance, oxidative stress resistance, and yield. In furtherembodiments, such treatments may induce a plant response generallytermed “Systemic acquired resistance” (“SAR”).

Dicamba, 3,6-DCSA, DCGA, and other products of DMO-mediated metabolismof dicamba, and substrates of DMO may thus induce improvements in planthealth. A “product of DMO-mediated metabolism of dicamba” may include3,6-DCSA, a product of metabolism of 3,6-DCSA, or an analog thereof.Proteins produced in response to application of dicamba or DCSA may havea direct antimicrobial activity (e.g. a pathogenesis-related proteinsuch as chitinase), or may have another function that potentiates one ormore of improved plant health, disease resistance, oxidative stressresistance, and enhanced yield. Thus, one aspect of the invention is amethod of producing 3,6-DCSA or DCGA comprising contacting a populationof plants in a crop or production field with dicamba. The plants may betransgenic plants comprising a DMO transgene.

In certain aspects of the invention, a plant may be contacted with aproduct of in planta DMO-mediated metabolism of dicamba, such as3,6-DCSA, or DCGA, such that the plant displays enhanced stressresistance as compared to an otherwise identical plant that has not beencontacted with dicamba or a product of DMO-mediated metabolism ofdicamba.

In one embodiment, the plant may be defined as lacking a DMO transgeneand contacted with a non-herbicidal product of DMO-mediated metabolismof dicamba, such as 3,6-DCSA. It was found, for example, that cottonplants contacted with 3,6-DCSA survive inoculation with the plantpathogenic oomycete Pythium ultimum that under the same conditions killsotherwise identical plants grown under the same conditions but notcontacted with 3,6-DCSA.

In another aspect, a plant may be contacted with dicamba, 3,6-DCSA,DCGA, and/or other products of DMO-mediated metabolism in planta, whichyields increased tolerance to oxidative stress. Such stress may beenvironmental or, for instance, be caused by the presence of anherbicide, pathogen, or other agent such as ozone. In one embodiment,the tolerance to oxidative stress is enhanced such that the plant'sphotosynthetic activity is not decreased, or is less affected, by thepresence of the oxidative stress. Photosynthetic activity may be assayedby means well known in the art, for instance, by measuring electrontransfer through photosystems I and/or II (e.g. Peterson and Arntzen,1982; Allen and Holmes, 1986). In another embodiment, the growth,development, flowering, or yield of the plant is not deleteriouslyaffected, or is less affected, by the presence of an oxidative stress ifthe plant has been contacted with dicamba, 3,6-DCSA, or another productof DMO-mediated metabolism of dicamba. In yet another embodiment, thenecrotizing effect of oxidative stress is reduced.

In specific embodiments of the invention, a plant treated in accordancewith the invention may be defined as growing in a crop production field.By “crop production field” is meant a growing environment in which acrop plant is typically grown in a field for production purposes,including seed production, rather than a laboratory greenhouse. Infurther embodiments of the invention, a population of plants may bedefined as growing in a crop production field and treated in accordancewith the invention. The plant treated in accordance with the inventionmay be an immature plant undergoing vegetative growth and sensitive todisease or weed pressure, such as a soybean plant in growth stage VE toV3-V4. The plant may also be at a later growth stage.

The chemical structure of 3,6-DCSA is as follows (I):

An analog of 3,6-DCSA may be defined, for example, as a substitutedbenzoic acid, and biologically acceptable salts thereof, whereinsubstitution on the benzoic acid may include mono-, di-, tri-, ortetra-substitution at the 3-, 4-, 5- and/or 6-positions. Substituentsmay be chosen for instance from among: lower alkyl groups of 1 to 4carbons; the halogens fluorine, chlorine, bromine or iodine; an aminogroup, wherein the nitrogen may carry 0, 1, or 2 identical or differentlower alkyl groups of from 1 to 4 carbons each; the nitro group; theformyl group; the acetyl group; the hydroxymethyl group; themethoxycarbonyl group; the hydroxyl group; an alkylthio-, alkylsulfoxyor alkylsulfonyl group, wherein the alkyl group is comprised of from 1to 4 carbons, or a mono-, di- or trifluoromethyl group. Biologicallyacceptable salts include those of the common alkali metals sodium andpotassium, the alkaline earths magnesium or calcium, zinc, or ammoniumor simple alkylammonium cations, such as mono-, di-, tri- ortetramethylammonium cations.

The product of DMO-mediated metabolism may be conjugated to a glucosidewhich may be hydrolyzed back to the aglycone to modulate, e.g. prolong,the health benefit. In certain embodiments, conjugation to a sugar suchas glucose, galactose, or mannose, among others, is contemplated. Inother embodiments, conjugation to an amino acid (non-limiting examplesof which include alanine, leucine, aspartate, or glutamate) iscontemplated.

In still another aspect, the application or presence of dicamba, or3,6-DCSA, or other product of DMO-mediated metabolism confers enhancedyield to a plant, as compared to the yield of the plant of the samegenotype not contacted with dicamba, or 3,6-DCSA, or other product ofDMO-mediated metabolism, but grown in the same conditions. In certainembodiments, the plant may be a soybean, cotton, rapeseed, or cornplant, among others.

In another aspect, a plant may be contacted with a non-herbicidalprecursor molecule that is converted to a SAR-inducing metabolite withinthe plant. In certain embodiments, the plant may comprise a DMOtransgene, and the resulting encoded DMO may convert the precursormolecule to a SAR-inducing metabolite. In particular embodiments,2-methoxy, 3,5-dichloro benzoic acid or 2-methoxy, 3-chloro benzoic acidmay be applied to a plant and converted by DMO to 3,5-DCSA, or 3-CSA,respectively, to yield any one or more of improved plant health,increased disease resistance of a plant, enhanced tolerance to oxidativestress on a plant, and/or enhancement of the agronomic yield of a plant.

The methods of the invention may be used in connection with, in oneembodiment, dicotyledonous (dicot) crop plants. Non-limiting examples ofsuch dicotyledonous plants include alfalfa, beans, beet, broccoli,cabbage, canola, carrot, cauliflower, celery, Chinese cabbage, cotton,cucumber, eggplant, lettuce, melon, pea, pepper, peanut, potato,pumpkin, radish, rapeseed, spinach, soybean, squash, sugarbeet,sunflower, tomato, and watermelon. In some embodiments, the dicot issoybean, cotton, or rapeseed. In other embodiments, the methods of theinvention may be used in connection with monocotyledonous crop plantsincluding, but not limited to, barley, corn, leek, onion, rice, sorghum,sweet corn, wheat, rye, millet, sugarcane, oat, triticale, switchgrass,and turfgrass.

Biotic and abiotic crop stress conditions may include, for example,drought, shade, fungal disease, viral disease, bacterial disease, insectinfestation, nematode infestation, weed infestation, cold temperatureexposure, heat exposure, osmotic stress, oxidative stress, reducednitrogen nutrient availability, reduced phosphorus nutrient availabilityand high plant density. Such conditions may be unfavorable for a plant,which adversely affects plant metabolism, growth and/or development.

The methods of the present invention find use in the control, preventionor treatment of a wide variety of plant diseases. The methods of thepresent invention include prophylactic inhibition and therapeutictreatment of infection by plant pathogens. Plant pathogens can beclassified by their life cycle in relation to a plant host, theseclassifications include, obligatge parasites, facultative parasites, andfacultative saprophytes. Obligate parasites can only survive andreproduce by obtaining nutrition from living plant cells and are indirect contact with these cells, examples of obligate fungal parasitesof plants include, but are not limited to members of Uredinales (rusts),Ustilaginales (smuts and bunts), Erysiphales (powdery mildews), andOomycetes (water molds and downy mildews), Facultative parasites areorganisms that generally survive as saprophytes on the products of otherorganisms or dead organisms but can become parasitic when the conditionsare favorable. Facultative saprophytes are organisms that generallysurvive as parasites of plants but can survive as saprophytes when asusceptible plant host is not available. The method of the presentinvention can be used to control, prevent or treat infection from a widearray of plant pathogens that include obligate parasites, facultativeparasites, and facultative saprophytes, which include, but are notlimited to the following:

Ascomycete fungi such as of the genera Venturia, Podosphaera, Erysiphe.Monilinia, Mycosphaerella, and Uncinula; Basidiomycete fungi such asfrom the genera Hemileia, Rhizoctonia, and Puccinia; Fungi imperfectisuch as the genera Botrytis, Helminthosporium, Rhynchosporiurn, Fusarium(i.e. F. monoliforme), Septoria, Cercospora, Alternaria, Pyricularia,Pseudocercosporella (i.e., P. herpotrichoides), and Verticillium;Oomycete fungi such as from the genera Phytophthora (i.e., P.parasitica, P. medicaginis, P. megasperma), Peronospora (i.e, P.tabacina), Bremia, Pythium, and Plasmopara; as well as other fungi suchas Scleropthora macrospora, Sclerophthora rayissiae, Sclerosporagraminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora sacchari and Peronosclerospora maydis, Physopellazeae, Cercospora zeae-maydis, Colletotrichum graminlicola. Gibberellazeae, Exserohilum turcicum, Kabatiella zeae, and Bipolaris maydis maycause disease that is controlled, prevented, or treated by the methodsof the present invention. Particularly preferred pathogens include, butare not limited to: Puccinia, Rhizoctonia, GOT (Gaeumannomyces graminisvar. tritici), stripe rust, Phakopsora sp. including P. pachyrhizi(causing Asian soybean rust), Fusarium species, Verticillium speciessuch as V. dahliae, Cercospora zeae-maydis (causing Gray leaf spot),Phytophthora species and Corn rust. Thus, the diseases controlled,prevented or treated include, for example, diseases of alfalfa plantssuch as root rot (Phytophora medicaginis, P. megasperma); rice plantsuch as Rice blast (Pyricularia oryzae), Helminthosporium leaf blight(Helminthosporium oryzae, Cochliobolus miyabeanus), Bakanae disease(Gibberella fujikuroi), Seedling blight (Rhizopus oryzae), Sheath blight(Rhizoctonia solani), and so on; those of oat such as Crown rust(Puccinia coronata), and so on; those of barley such as Powdery mildew(Erysiphe graminis), Scald (Rhynchosporium secalis), Spot-blotch(Cochliobolus sativus), Yellow mottleleaf (Helminthosporium gramineun,Pyrenophora gramineum), Net blotch (Pyrenophora teres), Stinking smut(Tilletia caries), Loose smut (Ustilago nuda), and so on; those of wheatsuch as Powdery mildew (Erysiphe graminis), Glume-blotch (Leptosphaerianodorum, Septoria nodorum), Stripe rust (Puccinia striiformis), Typhulasnow blight (Typhula incarnata), Eye spot (Pseudocercosporellaherpotrichoides), Snow mold (Calonectria graminicola, Fusarium nivale),Stem rust (Puccinia graminis), Black snow blight (Typhulaishikariensis), Scab (Gibberella zeae), Leaf rust (Puccinia recondita,Puccinia triticina), Stripe (Helminthosporium gramineum), Stinking smut(Tilletia caries), Speckled leaf blight (Septoria tritici), Loose smut(Ustilago tritici), and so on; those of turfgrass such as Gray leaf spot(Pyricularia grisea) and so on; those of corn such as Corn rust,Damping-off (Pythium debaryanum), and so on; those of rye such as Purplesnow mold (Fusarium nivale), and so on; those of cotton such asVerticillium wilt (V. dahliae), Seedling disease (Pythium ultimum,Colletotrichum gossypii, Pythium sp.; Rhizoctonia solani, Thielaviopsissp.), Bacterial blight (X. campestris pv. malvacearum), and so on; thoseof potato such as Late blight (Phytophthora infestans), and so on; thoseof tobacco plants such as Downy mildew (Peronospora tabacina), Foot rot(Phytophthora parasitica var), Septoria blight (Cercospora nicotianae),and so on; those of sugar beet such as Leaf spot (Cercospora beticola),Damping-off (Pythium debaryanum, Rhizoctonia solani, Pythiumaphanidermatum), and so on; those of paprika such as Gray mold (Botrytiscinerea), and so on, those of kidney bean such as Gray mold (Botrytiscinerea), Sclerotinia seed rot (sclerotial rot; Sclerotiniasclerotiorum), Southern blight (Corticium rolfsii), and so on; those ofbroad bean such as Powdery mildew (Erysiphe polygoni, Sphaerothecafuliginea), Rust (Uromyces fabae, Uromyces phaseoli), Gray mold(Botrytis cinerea), and so on; those of peanut such as Ascochyta spot(Mycosphaerella arachidicola), and so on; those of cabbage such asDamping blight (Rhizoctonia soloni), and so on; those of cucumber suchas Powdery mildew (Sphaerotheca fuliginea), Stem rot (Fusariumoxysporum), Gummy stem blight (Mycosphaerella melonis), Downy mildew(Pseudoperonospora cubensis), Gray mold (Botrytis cinerea), Sclerotialseed rot (Sclerotinia sclerotiorum), Anthracnose (Colletotrichumagenarium), Damping blight (Fusarium oxysporum, Pythium aphanidermatum,Rhizoctonia solani), and so on; those of Komatsuna (i.e. Brassica rapavar.) such as Alternaria sooty spot (Alternaria brassicicola), Club root(Plasmodiophora brassicae), and so on; those of celery such as Speckledleaf blotch (Septoria apii), and so on; those of radish such as Yellows(Fusarium oxysporum), and so on. those of tomato such as Fusarium wilt(Fusarium oxysporum), Foot rot (Phytophthora infestans), Ring leaf-spot(Alternaria solani), Gray mold (Botrytis cinerea), Leaf blight(Phytophthora capsici), Black rot (Alternaria tomato), and so on; thoseof eggplant such as Brown rot (Phytophthora capsici) vascular wiltpathogens, e.g. Verticillium wilt (Verticillium albo-atrum. V. dahliae),and so on, those of Chinese cabbage such as Black rot (Alternariajaponica), Club root (Plasmodiophora brassicae), and so on; those ofsweet pepper such as Foot rot (Phytophthora capsici), Gray mold(Botrytis cinerea), and so on; those of lettuce such as Gray mold(Botrytis cinerea), and so on, those of citrus fruits such as Pod andstem blight (Diaporthe citri), and so on, those of pear such as Scab(Venturia nashicola), Black rot (Alternaria kikuchiana). Japanese PearRust (Gymnosporangium haraeanum), Brown spot (caused by Stemphyliumvesicarium) and so on; those of grape such as Downy mildew (Plasmoparaviticola), Gray mold (Botryis cinerea), Sphaceloma scab (Elsinoëampelina), and so on; those of peach such as Leaf curl (Taphrinadeformans), Scab (Cladosporium carpophilum), shot hole (Mycosphaerellacerasella), and so on, those of apple such as Powdery mildew(Podosphaera leucotricha), Scab (Venturia inaequalis), Gray mold(Botrytis cinerea), Black rot (Botryosphaeria obtusa), Brown spot(Gymnosporangium yamadae), White root rot (Rosellinia necatrix),Alternaria leaf spot (Alternaria mali), and so on; and other diseases ofgrains, fruits and vegetables such as oil-seed rape, sunflower, carrot,pepper, strawberry. melon, kiwi fruit, onion, leek, sweet potato, fig,ume, asparagus, persimmon, soybean, adzukibean, watermelon, crown daisy,spinach, tea and so on.

Viral, bacterial, and nematode-caused diseases, such as viral mosaicdiseases (e.g. caused by Tobacco mosaic virus, Soybean mosaic virus,Alfalfa mosaic virus, or Cucumber mosaic virus), soybean dwarf virus,and bean pod mottle virus, among others: those caused by bacteriaincluding Pseudomonas syringae pvs. such as P. syringae pv. glycinea, P.svringae pv. coronafaciens: Xanthomonas campestris pvs., such as X.campestris pv. glycines; Xanlhomonas oryzae: Xanthomonas translucens;Xanthomonas axonopodis pv. malvacearum (Bacterial blight of cotton):Enrwinia spp. and including Pantoea spp.: and Clavibacter spp., amongothers, are also included, as well as those caused by nematodes such asMeloidogyne incognita, Pratylenchus penetrans, Xiphinema sp., andHeterodera sp. among others.

In one embodiment of the invention, the biotic crop stress is a rustfungus (Basidiomycete). Some agriculturally important plant rustdiseases include, without limitation, those caused by Puccinia sp., suchas cereal rusts caused by Puccinia coronata, Puccinia graminis, Pucciniastriiformis, Puccinia sorghi, Puccinia polysora, and P. recondita; rustscaused by Gymnosporangium sp.; White Pine Blister Rust caused byCronartium ribicola; Coffee Rust caused by Hemileia vastatrix; and rustdiseases caused by Uromyces sp. In a particular embodiment, the crop issoybean and the biotic crop stress is Soybean Rust caused by Phakopsorasp.

In certain aspects of the invention, dicamba or 3,6-DCSA may be providedor applied to a plant alone or in combination with another herbicide orother active ingredient. Application of the other herbicide may occurprior to, concurrently, or after application of the dicamba, 3,6-DCSA,or other product of DMO-mediated metabolism of dicamba.

“Application to a plant” may also comprise applying dicamba, 3,6-DCSA,or other product of DMO-mediated metabolism of dicamba, to a seed.“Health” of a seed may be measured, for instance, in terms of percentgermination, time to germination, resistance to seedling diseases orstresses, seedling vigor, or by the stand of a resulting crop.

The preparation of such compositions for use in connection with thecurrent invention will be apparent to those of skill in the art in viewof the disclosure. These compositions will typically include, inaddition to the active ingredient, components such as surfactants, solidor liquid carriers, solvents and binders. Examples of surfactants thatmay be used for application to plants include the alkali metal, alkalineearth metal or ammonium salts of aromatic sulfonic acids, e.g., ligno-,phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fattyacids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fattyalcohol sulfates and of fatty alcohol glycol ether sulfates, condensatesof sulfonated naphthalene and its derivatives with formaldehyde,condensates of naphthalene or of the naphthalenesulfonic acids withphenol and formaldehyde, condensates of phenol or phenolsulfonic acidwith formaldehyde, condensates of phenol with formaldehyde and sodiumsulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-,octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylarylpolyether alcohols, isotridecyl alcohol, ethoxylated castor oil,ethoxylated triarylphenols, salts of phosphatedtriarylphenolethoxylates, lauryl alcohol polyglycol ether acetate,sorbitol esters, lignin-sulfite waste liquors or methylcellulose, ormixtures of these. Common practice in the case of surfactant use isabout 0.25% to 1.0% by weight, and more commonly about 0.25% to 0.5% byweight.

In one embodiment of the invention, dicamba may be provided incombination with glyphosate. By “in combination with” it is meant thatdicamba may be applied concurrently, or prior or after glyphosate. Dueto synergism, this embodiment may be used to reduce amounts of eitherherbicide to achieve the same degree of activity as an application ofonly one of the herbicides. For example, the invention may involveapplying less than a 1× rate of glyphosate and/or dicamba, relative tothe standard manufacturer labeled rate. Examples of respectiveglyphosate and dicamba application rates include, in addition to 1×rates, from about a 0.25×-0.95× of either herbicide, specificallyincluding about 0.5×, 0.6×, 0.7×, 0.8×. 0.85×, 0.9×, and 0.95× of eitherherbicide and all derivable combinations thereof, as well as higherrates such as 0.97× and 0.99×. In certain plant health embodiments, itmay be desirable to use individual applications of dicamba in theseamounts. Alternatively, 1× and higher application rates may be made inview of the finding that even high application rates of dicamba do notsignificantly damage plants containing a DMO transgene. The 1×application rates are set by the manufacturer of a commerciallyavailable herbicide formulation and are known to those of skill in theart. For example, the label for Fallow Master™, a glyphosate and dicambamixture having a ratio of glyphosate:dicamba of about 2:1 recommendsapplication rates of about 451 g/ha (311 ae g/ha glyphosate:140 ae g/hadicamba) to 621 ae g/ha (428 ae g/ha glyphosate: 193 ae g/ha dicamba)depending upon the weed species and weed height. Glyphosate may also beapplied in combination with 3,6-DCSA. Beneficial effects on plant healthmay be obtained by contacting plants or plant parts with dicamba byutilizing dicamba for weed control. Or, a non-herbicidal rate of dicambamay be provided to a crop, which nevertheless results in beneficialhealth effects.

The dicamba, or 3,6-DCSA, or analog of 3,6-DCSA, or product thereof thatcan be produced via DMO-mediated metabolism of dicamba, may be appliedoutside of a typical application window for weed control, by forinstance varying the timing of application, the growth stage of the cropand/or weed plants, or the application rate, according to the knowledgeof one of skill in the art. The plant identified as in need of improvedhealth may be an immature plant undergoing vegetative growth andsensitive to a disease or to weed growth, such as a soybean plant ingrowth stage VE to V3 or V4. The soybean plant may also be at a post-V4growth stage. The plant may also be a corn plant prior to or duringgrowth stages such as VE to V3, V7, V10, V15, or VT. The corn plant mayalso be at a post-vegetive growth stage. The plant may also be a cottonplant undergoing vegetative growth such as prior to blooming, includingfor instance, prior to or during the seedling emergence, first trueleaf, first vegetative side shoot, first fruiting branch, or 1^(st)square (fruiting bud) formation growth stages. The cotton plant may alsobe at a flowering growth stage, or during boll-development. The growthstage of a plant may also be defined by days after planting.

“Dicamba” refers to 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid and its acids and salts. Its salts include isopropylamine,diglycoamine, dimethylamine, potassium and sodium. Examples ofcommercial formulations of dicamba include, without limitation, Banvel™(as DMA salt), Clarity® (as DGA salt), VEL-58-CS-11™ and Vanquish™ (asDGA salt, BASF). “Glyphosate” refers to N-phosphonomethylglycine andsalts thereof. Glyphosate is commercially available in numerousformulations. Examples of these formulations of glyphosate include,without limitation, those sold by Monsanto Company as ROUNDUP®, ROUNDUP®ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP®BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD®herbicides, all of which contain glyphosate as its isopropylammoniumsalt, ROUNDUP® WEATHERMAX containing glyphosate as its potassium salt;ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as itsammonium salt; ROUNDUP®GEOFORCE, which contains glyphosate as its sodiumsalt; and TOUCHDOWN® herbicide, which contains glyphosate as itstrimethylsulfonium salt.

In certain embodiments, the invention may relate to use of a DMOtransgene. In one aspect of the invention, the DMO may be encoded by anucleic acid sequence selected from the group consisting of: a) anucleic acid sequence encoding the polypeptide of SEQ ID NO:1; b) anucleic acid sequence comprising the sequence of SEQ ID NO:2; and c) anucleic acid sequence encoding a polypeptide with at least 90% sequenceidentity to the polypeptide of SEQ ID NO:1 wherein the polypeptide hasdicamba monooxygenase activity and comprises cysteine at a positioncorresponding to amino acid 112 of SEQ ID NO:1. In another aspect, thenucleic acid sequence may encode a polypeptide with at least 90%sequence identity to the polypeptide of SEQ ID NO:1 that has dicambamonooxygenase activity and comprises tryptophan at a positioncorresponding to amino acid 112 of SEQ ID NO:1. In such embodiments, aDNA vector may be provided comprising a DMO-encoding nucleic aciddescribed herein operably linked to a promoter. The promoter may befunctional in a plant cell. In certain embodiments, the nucleic acidsequence encoding dicamba monooxygenase may be operably linked to achloroplast transit peptide (CTP). In other embodiments, the inventionmay relate to use of a transgene that confers tolerance to glyphosate,such as CP4 EPSPS. A sequence of such a gene may be found, for example,in U.S. Pat. RE39,247, herein incorporated by reference.

DMOs having a capability to degrade dicamba, as well as glyphosate- orother herbicide-tolerance genes, can readily be prepared and assayed foractivity according to standard methods. Such sequences can also beidentified by techniques known in the art, for example, from suitableorganisms including bacteria that degrade herbicides (U.S. Pat. No.5,445,962; Cork and Krueger, 1991; Cork and Khalil, 1995). One means ofisolating a DMO or other sequence is by nucleic acid hybridization, forexample, to a library constructed from the source organism, or by RT-PCRusing mRNA from the source organism and primers based on the disclosedsequences. The invention therefore encompasses use of nucleic acidshybridizing under stringent conditions to a DMO encoding sequencedescribed herein. One of skill in the art understands that conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results. An example of high stringency conditions is 5×SSC,50% formamide and 42° C. By conducting a wash under such conditions, forexample, for 10 minutes, those sequences not hybridizing to a particulartarget sequence under these conditions can be removed.

Variants can also be chemically synthesized, for example, using theknown DMO polynucleotide sequences according to techniques well known inthe art. For instance, DNA sequences may be synthesized byphosphoroamidite chemistry in an automated DNA synthesizer. Chemicalsynthesis has a number of advantages. In particular, chemical synthesisis desirable because codons preferred by the host in which the DNAsequence will be expressed may be used to optimize expression. Not allof the codons need to be altered to obtain improved expression, butpreferably at least the codons rarely used in the host are changed tohost-preferred codons. High levels of expression can be obtained bychanging codons to host-preferred codons. The codon preferences of manyhost cells are known (PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP553494; and U.S. Pat. Nos. 5,689,052; 5,567,862; 5,567,600; 5,552,299and 5,017,692). The codon preferences of other host cells can be deducedby methods known in the art. Also, using chemical synthesis, thesequence of the DNA molecule or its encoded protein can be readilychanged to, for example, optimize expression (for example, eliminatemRNA secondary structures that interfere with transcription ortranslation), add unique restriction sites at convenient points, anddelete protease cleavage sites.

Modification and changes may be made to the polypeptide sequence of aprotein such as the DMO sequences provided herein while retainingenzymatic activity. The following is a discussion based upon changingthe amino acids of a protein to create an equivalent, or even animproved, modified polypeptide and corresponding coding sequences. It isknown, for example, that certain amino acids may be substituted forother amino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as binding sites onsubstrate molecules. Since it is the interactive capacity and nature ofa protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence, and, of course, its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated that various changes may be made in the DMO peptidesequences described herein or other herbicide tolerance polypeptides andcorresponding DNA coding sequences without appreciable loss of theirbiological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte et al., 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics (Kyte et al., 1982), theseare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is known in the art that amino acids may be substituted by otheramino acids having a similar hydropathic index or score and still resultin a protein with similar biological activity, i.e., still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent protein.In such changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred. Exemplary substitutions which take these and various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

A DNA construct comprising a CTP sequence operably linked to a DMOsequence can be expressed in test system such as protoplasts,transiently or stably transformed plant cells by operably linked them toa promoter functional in plants. Examples describing such promotersinclude U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No.5,641,876 (rice actin promoter; OsAct1), U.S. Pat. No. 6,426,446 (maizeRS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S.Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611(constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605,5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maizeL3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter aswell as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specificpromoter), U.S. Pat. No. 6,294,714 (light inducible promoters), U.S.Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138(pathogen inducible promoters), U.S. Pat. No. 6,175,060 (phosphorusdeficiency inducible promoters), U.S. Pat. No. 6,388,170 (e.g. PClSVpromoter), the PClSV promoter of SEQ ID NO:41, U.S. Pat. No. 6,635,806(gamma-coixin promoter), and U.S. Pat. No. 7,151,204 (maize chloroplastaldolase promoter). Additional promoters that may find use are anopaline synthase (NOS) promoter (Ebert et al., 1987), the octopinesynthase (OCS) promoter (which is carried on tumor-inducing plasmids ofAgrobacterium tumefaciens), the caulimovirus promoters such as thecauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), theCaMV 35S promoter (Odell et al., 1985), the figwort mosaic virus35S-promoter (Walker et al., 1987), the sucrose synthase promoter (Yanget al., 1990), the R gene complex promoter (Chandler et al., 1989), andthe chlorophyll a/b binding protein gene promoter, etc. In the presentinvention, CaMV35S with enhancer sequences (e35S; U.S. Pat. Nos.5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Pat. Nos.6,051,753; 5,378,619), peanut chlorotic streak caulimovirus (PClSV; U.S.Pat. No. 5,850,019), At.Act 7 (Accession # U27811), At.ANT1 (US PatentApplication 20060236420), FMV.35S-EF1a (US Patent Application20050022261), eIF4A10 (Accession #X79008) and AGRtu.nos (GenBankAccession V00087; Depicker et al, 1982; Bevan et al., 1983), ricecytosolic triose phosphate isomerase (OsTPI; U.S. Pat. No. 7,132,528),and rice actin 15 gene (OsAct15; U.S. Patent Application 2006-0162010)promoters may be of particular benefit.

A 5′ UTR that functions as a translation leader sequence is a DNAgenetic element located between the promoter sequence of a gene and thecoding sequence may be included between a promoter and CTP-DMO sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences include maize and petunia heat shock protein leaders (U.S.Pat. No. 5,362,865), plant virus coat protein leaders, plant rubiscoleaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No.5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), and AGRtunos(GenBank Accession V00087; Bevan et al., 1983) among others. (Turner andFoster, 1995). In the present invention, 5′UTRs that may in particularfind benefit are GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No.5,362,865), AtAnt 1, TEV (Carrington and Freed, 1990), OsAct1 (U.S. Pat.No. 5,641,876), OsTPI (U.S. Pat. No. 7,132,528), 0 sAc t15 (U.S.Publication No. 20060162010), and AGRtunos (GenBank Accession V00087;Bevan et al., 1983).

The 3′ non-translated sequence, 3′ transcription termination region, orpoly adenylation region means a DNA molecule linked to and locateddownstream of a structural polynucleotide molecule and includespolynucleotides that provide polyadenylation signal and other regulatorysignals capable of affecting transcription, mRNA processing or geneexpression. The polyadenylation signal functions in plants to cause theaddition of polyadenylate nucleotides to the 3′ end of the mRNAprecursor. The polyadenylation sequence can be derived from the naturalgene, from a variety of plant genes, or from T-DNA genes. Thesesequences may be included downstream of a CTP-DMO sequence. An exampleof a 3′ transcription termination region is the nopaline synthase 3′region (nos 3′; Fraley et al., 1983). The use of different 3′nontranslated regions is exemplified (Ingelbrecht et al., 1989).Polyadenylation molecules from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9;Coruzzi et al., 1984), AGRtu.nos (Genbank Accession E01312), E6(Accession #U30508), and TaHsp17 (wheat low molecular weight heat shockprotein gene; Accession #X13431) in particular may be of benefit for usewith the invention.

In addition to expression elements described above, an intron may berequired in between a promoter and a 3′ UTR for expressing a codingregion, especially in monocots. An “intron” refers to a polynucleotidemolecule that may be isolated or identified from the interveningsequence of a genomic copy of a gene and may be defined generally as aregion spliced out during mRNA processing prior to translation.Alternately, introns may be synthetically produced. Introns maythemselves contain sub-elements such as cis-elements or enhancer domainsthat effect the transcription of operably linked genes. A “plant intron”is a native or non-native intron that is functional in plant cells. Aplant intron may be used as a regulatory element for modulatingexpression of an operably linked gene or genes. A polynucleotidemolecule sequence in a transformation construct may comprise introns.The introns may be heterologous with respect to the transcribablepolynucleotide molecule sequence. Examples of introns include the cornactin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron (ZmHSP70;U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and rice TPI intron(OsTPI; U.S. Pat. No. 7,132,528) and are of benefit in practicing thisinvention.

Any of the techniques known in the art for introduction of transgeneconstructs into plants may be used in accordance with the invention(see, for example, Mild et al., 1993). Suitable methods fortransformation of plants are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by electroporation asillustrated in U.S. Pat. No. 5,384,253; microprojectile bombardment asillustrated in U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;6,160,208; 6,399,861; and 6,403,865; Agrobacterium-mediatedtransformation as illustrated in U.S. Pat. Nos. 5,635,055; 5,824,877;5,591,616; 5,981,840; and 6,384,301; and protoplast transformation asillustrated in U.S. Pat. No. 5,508,184. Through the application oftechniques such as these, the cells of virtually any plant species maybe stably transformed, and these cells may be developed into transgenicplants. Techniques that may be particularly useful in the context ofcotton transformation are disclosed in U.S. Pat. Nos. 5,846,797,5,159,135, 5,004,863, and 6,624,344. Techniques for transformingBrassica plants in particular are disclosed, for example, in U.S. Pat.No. 5,750,871; and techniques for transforming soybean are disclosed in,for example, Zhang et al., 1999, U.S. Pat. No. 6,384,301, and U.S. Pat.No. 7,002,058. Techniques for transforming corn are disclosed, forinstance, in WO9506722. Some non-limiting examples of plants that mayfind use with the invention include alfalfa, barley, beans, beet,broccoli, cabbage, carrot, canola, cauliflower, celery, Chinese cabbage,corn, cotton, cucumber, dry bean, eggplant, fennel, garden beans, gourd,leek, lettuce, melon, oat, okra, onion, pea, pepper, pumpkin, peanut,potato, pumpkin, radish, rice, sorghum, soybean, spinach, squash, sweetcorn, sugarbeet, sunflower, switchgrass, tomato, watermelon, and wheat.

After effecting delivery of exogenous DNA to recipient cells, the nextsteps in generating transgenic plants generally concern identifying thetransformed cells for further culturing and plant regeneration. In orderto improve the ability to identify transformants, one may desire toemploy a selectable or screenable marker gene with a transformationvector prepared in accordance with the invention. In this case, onewould then generally assay the potentially transformed cell populationby exposing the cells to a selective agent or agents, or one wouldscreen the cells for the desired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. Any suitable plant tissue culturemedia, for example, MS or N6 media (Murashige and Skoog, 1962; Chu etal., 1975); may be modified by including further substances such asgrowth regulators. Tissue may be maintained on a basic media with growthregulators until sufficient tissue is available to begin plantregeneration efforts, or following repeated rounds of manual selection,until the morphology of the tissue is suitable for regeneration,typically at least 2 weeks, then transferred to media conducive to shootformation. Cultures are transferred periodically until sufficient shootformation had occurred. Once shoot are formed, they are transferred tomedia conducive to root formation. Once sufficient roots are formed,plants can be transferred to soil for further growth and maturity.

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR™; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

Once a transgene has been introduced into a plant, that gene can beintroduced into any plant sexually compatible with the first plant bycrossing, without the need for ever directly transforming the secondplant. Therefore, as used herein the term “progeny” denotes theoffspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. A “transgenicplant” may thus be of any generation. “Crossing” a plant to provide aplant line having one or more added transgenes or alleles relative to astarting plant line, as disclosed herein, is defined as the techniquesthat result in a particular sequence being introduced into a plant lineby crossing a starting line with a donor plant line that comprises atransgene or allele of the invention. To achieve this one could, forexample, perform the following steps: (a) plant seeds of the first(starting line) and second (donor plant line that comprises a desiredtransgene or allele) parent plants; (b) grow the seeds of the first andsecond parent plants into plants that bear flowers; (c) pollinate aflower from the first parent plant with pollen from the second parentplant; and (d) harvest seeds produced on the parent plant bearing thefertilized flower.

Unmodified and modified protein molecules and their correspondingnucleic acid molecules providing tolerance to one or more herbicides arewell known in the art. For example:

a) sequences encoding tolerance to glyphosate include5-enolpyruvylshikimate-3-phosphate synthases (EPSPS; U.S. Pat. No.5,627,061, U.S. Pat. RE39,247, U.S. Pat. No. 6,040,497, U.S. Pat. No.5,094,945, WO04074443, and WO04009761), glyphosate oxidoreductase (GOX;U.S. Pat. No. 5,463,175), glyphosate decarboxylase (WO05003362 and U.S.Patent Application 20040177399), and glyphosate-N-acetyl transferase(GAT; U.S. Patent publication 20030083480) conferring tolerance toglyphosate;

b) dicamba monooxygenase (DMO, encoded by ddmC) conferring tolerance toauxin-like herbicides such as dicamba (U.S. Patent Applicationpublications 20030115626, 20030135879; Wang et al., 1996; Herman et al.,2005);

c) phosphinothricin acetyltransferase (bar) conferring tolerance tophosphinothricin or glufosinate (U.S. Pat. No. 5,646,024, U.S. Pat. No.5,561,236, EP 275,957; U.S. Pat. No. 5,276,268; U.S. Pat. No. 5,637,489;U.S. Pat. No. 5,273,894);

d) 2,2-dichloropropionic acid dehalogenase conferring tolerance to2,2-dichloropropionic acid (Dalapon) (WO9927116);

e) acetohydroxyacid synthase or acetolactate synthase conferringtolerance to acetolactate synthase inhibitors such as sulfonylurea,imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide(U.S. Pat. No. 6,225,105; U.S. Pat. No. 5,767,366, U.S. Pat. No.4,761,373; U.S. Pat. No. 5,633,437; U.S. Pat. No. 6,613,963; U.S. Pat.No. 5,013,659; U.S. Pat. No. 5,141,870; U.S. Pat. No. 5,378,824; U.S.Pat. No. 5,605,011);

f) haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil(WO8704181A1; U.S. Pat. No. 4,810,648; WO8900193A);

g) modified acetyl-coenzyme A carboxylase for conferring tolerance tocyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop)(U.S. Pat. No. 6,414,222);

h) dihydropteroate synthase (sulI) for conferring tolerance tosulfonamide herbicides (U.S. Pat. No. 5,597,717; U.S. Pat. No.5,633,444; U.S. Pat. No. 5,719,046);

i) 32 kD photosystem II polypeptide (psbA) for conferring tolerance totriazine herbicides (Hirschberg et al., 1983);

j) anthranilate synthase for conferring tolerance to 5-methyltryptophan(U.S. Pat. No. 4,581,847);

k) dihydrodipicolinic acid synthase (dapA) for conferring to toleranceto aminoethyl cysteine (WO8911789);

l) phytoene desaturase (crtl) for conferring tolerance to pyridazinoneherbicides such as norflurazon (JP06343473);

m) hydroxy-phenyl pyruvate dioxygenase for conferring tolerance tocyclopropylisoxazole herbicides such as isoxaflutole (WO 9638567; U.S.Pat. No. 6,268,549);

n) modified protoporphyrinogen oxidase I (protox) for conferringtolerance to protoporphyrinogen oxidase inhibitors (U.S. Pat. No.5,939,602); and

o) aryloxyalkanoate dioxygenase (AAD-1) for conferring tolerance to anherbicide containing an aryloxyalkanoate moiety (WO05107437). Examplesof such herbicides include phenoxy auxins (such as 2,4-D anddichlorprop), pyridyloxy auxins (such as fluoroxypyr and triclopyr),aryloxyphenoxypropionates (AOPP) acetyl-coenzyme A carboxylase (ACCase)inhibitors (such as haloxyfop, quizalofop, and diclofop), and5-substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors(such as pyraflufen and flumiclorac).

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Production of Transgenic Soybean Containing a DMO Gene

Transgenic soybean plants comprising a DMO-encoding transgene wereobtained by Agrobacterium-mediated transformation of soybean tissue fromcultivars Thorne, NE3001, and A3525, using standard procedures (e.g.U.S. Pat. No. 6,384,301, U.S. 7,002,058 or Zhang et al., 1999) with abinary vector containing a DMO-encoding polynucleotide which encodes thepolypeptide of SEQ ID NO:1.

Example 2 Induction of a Pathogen Related Protein Involved in DiseaseResistance by Dicamba and DCSA

Transgenic and control soybean seeds (cultivars Thorne, NE3001, A3525)were planted into 3.5-inch square plastic pots containing Redi-earth™(Scotts-Sierra Horticultural Products Co., Marysville, Ohio). The potswere placed on capillary matting in 35 inch×60 inch fiberglass wateringtrays for overhead and/or sub-irrigation for the duration of the testperiod so as to maintain optimum soil moisture for plant growth and werefertilized with Osmocote® (14-14-14 slow release; Scotts-SierraHorticultural Products Co., Marysville, Ohio) at the rate of 100gm/cu·ft. to sustain plant growth for the duration of greenhouse trials.

The plants were grown in greenhouses at 29°/21° C. day/night temperaturewith relative humidity between 15%-50% to simulate warm season growingconditions of late spring. A 14 hour minimum photoperiod was providedwith supplemental light at about 600 μE (micro-Einsteins) as needed.Trials were established in a randomized block design randomized by ratewith 4 to 6 replications of each treatment.

All herbicide applications were made with the track sprayer using aTeejet® 9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.) withair pressure set at a minimum of 24 psi (pounds per square inch), or 165kpa (kilopascals)). The spray nozzle was kept at a height of about 16inches above the top of plant material for spraying. The spray volumewas 10 gallons per acre or 93 liters per hectare. Applications were madewhen plants had reached V-3 stage. Events carrying a DMO transgene intheir genome were grown and treated with dicamba (Clarity®, BASF) at 1lb/Acre at Post-V3 stage. Leaf samples were harvested after 0, 3, 8, 24,48, 72 hrs after treatment (HAT) and frozen for further analysis. Inanother experiment, DMO-containing plants were sprayed with eitherdicamba or DCSA at 0.25, 0.5 and 1 lb/Acre rate and tissue samples werecollected 24 HAT. RNA was extracted and analyzed by northern blotanalysis using PR-2, i.e., β-1,3-glucanase as a probe. PR-2 is apathogenesis-related protein known to degrade fungal membranes and thusprovide protection against fungal pathogens (U.S. Pat. No. 5,670,706 andUknes et al., 1992).

Tissue samples were ground in Falcon™ tubes (BD Biosciences, FranklinLakes, N.J.) with liquid nitrogen and glass/metal beads. Sub-sampleswere taken in Eppendorf tubes. A plant RNA mini kit was used forextracting RNA following manufacturer protocol (Qiagen, Valencia,Calif.). RNA concentration was estimated using measurement of OD at 260and 280 nm. Five μg (micrograms) of RNA was precipitated by adding 1/10volume of 3M sodium acetate and 2.5 volume of 100% ethanol and storingthe tubes at −20° C. for 48 hrs. The tubes were centrifuged for 20 minat 4° C. and supernatant was discarded. Seventy percent ethanol wasadded to the tubes and RNA was resuspended in it gently. The tubes werethen centrifuged for 3.5 minutes, and the RNA pellet was dried for about30 to 60 min and resuspended in 15 μl of loading buffer (Gel loadingbuffer II from Ambion, Austin, Tex.) by brief vortexing and mixing.

RNA samples and RNA markers (RNA ladder; Invitrogen, Carlsbad, Calif.)were denatured at 65° C. for 10 min and kept on ice until loaded in gelmade from 1% agarose and 2% formaldehyde in 1×MOPS buffer. The gel wasrun at 17 V for 16 h in 1×MOPS buffer and stained with ethidium bromideto visualize RNA samples and markers in the gel. The gel was washed for30 min in water to remove ethidium bromide followed by two washes in20×SSC (NaCl and sodium citrate) for 15 min each.

RNA samples and markers were blotted on the membrane (Nytran® PLUS,Midwest Scientific, Valley Park, Mo.) using a rapid downward transfersystem (Turblotter™ and blotting stack, Whatman-Schleicher & Schuell;Florham Park, N.J.) for 5-6 hours. The membrane was rinsed with water,U.V. cross linked and placed on a paper to dry. The membrane waspre-hybridized in warm 15 ml DIG Easy Hyb buffer (Roche, Penzberg,Germany) at 50° C. for 30 min. PR-2 probe was labeled with DIG andsynthesized using PCR DIG probe synthesis kit according tomanufacturer's instructions (Roche) and boiled for 5 minutes and cooledon ice before adding to warm 15 μl DIG Easy Hyb buffer. Hybridizationwas done overnight at 50° C. The RNA blot was washed at high stringencytwice, for 5 min and 15 min, in 2×SSC and 0.1% SDS solution at roomtemperature. Two more washes followed for 30 min each in 0.5×SSC and0.1% SDS solution at 68° C. The blot was then washed in 1× washingbuffer (Roche) for 2 min and blocked using a blocking reagent (Roche)for 30 min to 3 hours at room temperature with moderate shaking. Theblot was transferred to 30 ml DIG antibody solution (75mU/ml, Roche) for30 min. Unbound antibodies were removed by 3 washes of 15 min each with1× washing buffer. The blot was equilibrated in 1× detection buffer(Roche) for 3 min. Ready to use CDP-STAR (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decan}-4-yl)-1-phenylphosphate; Roche) was added to the blot placed in a detection bag. Thebag was sealed and blot was exposed to BIOMAX film (Kodak) for 1.5hours.

FIG. 1 shows induction of PR2 at various time points after treating withdicamba. The peak induction was at 48 hrs. There was no induction of PR2in plants not treated with dicamba.

FIG. 2 shows induction of PR2 at various rates of dicamba and DCSA 24hrs after treatment in two independent transgenic lines of soybean.There was no induction of PR2 in plants not treated with dicamba or DCSAor treated with water only.

Induction of PR5 by dicamba or DCSA was also observed using a probespecific to PR5 (Uknes et al., 1992) and experimental procedures asdescribed above for PR2.

Example 3 DCSA Provides Fungal Disease Resistance

Ten-day old Roundup Ready® Flex Cotton (Event MON 88913; U.S. Publ.20060059590) seedlings were sprayed with a mixture of Roundup WeatherMax® (Monsanto Company, St. Louis, Mo.) and 3,6-DCSA (BASF). The controlplants were sprayed with water only. The cotton seedlings from bothtreatments were inoculated with Pythium ultimum by dipping seedling intoinoculum slurry, 24 hours after the spray application. The positivecontrol plants were neither sprayed nor inoculated with P. ultimum. Asshown in FIG. 3, the cotton seedlings inoculated with P. ultimum butthat did not receive application of a mixture of Roundup Weather Max®(Monsanto Company, St. Louis, Mo.) and 3,6-DCSA died as a result of P.ultimum infection within 10 days after inoculation. However, infectionby P. ultimum was not established in the plants that received a mixtureof Roundup Weather Max® (Monsanto Company, St. Louis, Mo.) and 3,6-DCSA.Cotton plants treated with Roundup® and DCSA were as healthy as were theplants not inoculated with P. ultimum. The results indicate that thecombined treatment of Roundup Ultra Max and DCSA reduced P. ultimuminfection when plants were inoculated with Pythium 24 hours after thetreatment with DCSA.

Example 4 DCSA Provides Bacterial Disease Resistance

In this example, two-week old cotton plants were sprayed with DCSA whilecontrol plants received spray application of water only. The plants wereinoculated with bacterial blight pathogen (Xanthomonas campestris pv.malvacearum) about 24 hours after the DCSA spray application using atooth pick inoculation method. As shown in the FIG. 4, the cotton plantsinoculated with the bacterial pathogen but not sprayed with DCSAdeveloped necrotic spots, typical symptoms of bacterial blight. However,the cotton plants that received DCSA application 24 hours beforeinoculation with bacterial pathogen showed only localized lesions (HRresponse) and no increase in bacterial blight symptoms. The results ofthis experiment indicate that DCSA application reduced bacterialinfection on cotton plants when inoculated with bacterial pathogen 24hours after the DCSA spray.

Example 5 Dicamba or DCSA Increases Tolerance to Oxidative Stress inSoybean

For this example, the plants were grown as described in the aboveexample. Transgenic and non transgenic soybean plants carrying the DMOgene in their genome were either treated with Paraquat (Gramoxone®;Syngenta) to create oxidative stress or with dicamba followed byParaquat at V3 stage. The dicamba rate was 1 lb/Acre or 1120 g ae/ha.Paraquat was applied at 30, 70, or 200 g ae/ha. Plants were thenevaluated for paraquat injury by visually assessing injury at aparticular day after treatment (DAT) for injury on a scale of 0 to 100percent relative to untreated control plants, with zero representing“no” injury and 100% representing “complete” injury or death. Data werecollected and analyzed using suitable statistical methods.

Table 1 shows that significantly reduced Paraquat injury was seen 4 DATat all three application rates tested on soybean plants carrying DMOgene and treated with dicamba 24 hrs before applying Paraquat.

TABLE 1 Percentage injury to non-transgenic or transgenic soybean plantstreated with paraquat or dicamba followed by paraquat at V-3 stage. The% injury was represented as ANOVA mean comparisons (with respect toparaquat treatment). Similar letters represent no statistical differenceat the p = 0.05 level. Paraquat (g ae/ha) Plant Treatment 30 g 70 g 200g N3001 Paraquat 32.5 a 50.8 a 75 a DMO-soy 462 Paraquat 33.3 a 48.3 a74.2 a DMO-soy 462 Dicamba f/b 16.7 b 26.7 b 51.7 b Paraquat DMO-soy 469Paraquat 30.8 a 49.2 a 76.7 a DMO-soy 469 Dicamba f/b 15 b 29.2 b 50 bParaquat LSD 6 6.7 6.3

In another experiment, the effect of externally applied DCSA was testedfor its benefit in protecting soybean plants from oxidative stressinjury caused by Paraquat. Different amounts of DCSA were applied 24 hrsbefore applying Paraquat to test any correlation between the amount ofDCSA applied and the extent of protection obtained from oxidative stressmeasured in terms of reduction in % injury as described above. Differentlevels of dicamba were also applied separately to test the samehypothesis.

Table 2 shows, for example, that the application of either dicamba orDSCA provided protection against oxidative stress caused by subsequentlyapplying paraquat. Transgenic plants contacted with Paraquat aloneexhibited an injury rate of about 75% whereas transgenic plantscontacted with different rates of dicamba or DSCA followed by Paraquatsurprisingly showed reduction in injury. Further, the reduction ininjury increased with increasing application rate of either dicamba orDCSA. The plants used in this experiment were transgenic. The effect ofdicamba is due to in planta conversion of dicamba into DCSA orsubsequent metabolism, whereas the effect of DCSA is due to DCSA itselfor due to its metabolites.

TABLE 2 Percentage injury to transgenic soybean plants treated withparaquat only, or with dicamba or DCSA followed by paraquat at V-3stage. The % injury was represented as ANOVA mean comparisons. % injuryat 5 DAT % injury at at different rates 5 DAT of Dicamba or DCSAParaquat followed by 100 g alone ae/ha of paraquat Transgenic control280 561 1120 Event Treatment 100 g ae/ha g/ha g/ha g/ha 462-1-21 Dicamba75.83 65.00 54.17 45.83 462-1-21 DCSA 75.83 66.67 53.33 49.17 469-13-19Dicamba 77.50 51.67 45.83 40.83 469-13-19 DCSA 77.50 50.83 48.33 43.33

Example 6 DCSA Increases Tolerance to Oxidative Stress in Cotton

Four different cotton cultivars as indicated in Table 3 were grown in4-inch pots for two weeks. These cotton plants were either sprayed withDCSA (BASF) at the 1 lb/A rate, or unsprayed (no DCSA application, ascontrol). Experimental plants (treated with DCSA and untreated) weresprayed with Paraquat (Gramoxone®; Syngenta) at 30 gm/ha, 70 gm/ha, and100 gm/ha rates, 24 hours after the application of DCSA, to simulateoxidative stress. The positive control plants did not receive anychemical treatment (neither DCSA nor Gramoxone® application). All plantswere then evaluated for paraquat injury by visual assessment two daysafter the treatment (DAT) with Gramoxone® on a scale of 0 to 100 percentrelative to untreated control plants, with zero representing “no” injuryand 100% representing “complete” injury or death of the plant. The sameexperiment was repeated at two different growth stages of cotton (2nodes stage and 5 nodes stage).

The results of these experiments shown in Table 3 indicate thatGramoxone® injury was reduced significantly when four varieties of RRcotton were pretreated with DCSA (24 hours) at two different growthstages (two nodes stage and 5 nodes stage). Reduction of injury symptomswas not dependent on the two cotton growth stages tested. Similarresults were obtained with non-transgenic cotton cultivar ST474.

TABLE 3 Percentage injury to indicated cotton plant varieties treatedwith paraquat or DCSA followed by paraquat. ST4646B2RF ST3273B2RFST4575BR ST6622RF UNTREATED 0 0 0 0 CONTROL Gramoxone ® 31.5 35 37.542.5 (30 g a.i./ha) DCSA (1 lb/Acre) + 8.5 6 12.5 7.5 Gramoxone ® (30 ga.i./ha) Gramoxone ® 42.5 50 42.5 50 (70 g a.i./ha) DCSA (1 lb/Acre) +20 32.5 30 35 Gramoxone ® (70 g a.i./ha) Gramoxone ® 70 50 65 70 (100 ga.i./ha) DCSA (1 lb/Acre) + 45 45 40 50 Gramoxone ® (100 g a.i./ha)

Example 7 Enhancing Yield by Application of Dicamba to Plants

The yield enhancing-benefit of DSCA was tested by growing non-transgenicand transgenic plants and applying dicamba to transgenic plants andharvesting the seeds. Non-transgenic and transgenic soybean seeds wereplanted near the beginning of the growing season in several locations atthe time of best possible growth conditions such as soil moisture,temperature, and seeding depth. Across all locations seeds were plantedunder split-plot design with dicamba treatments as whole-plot effectsand events as split-plot effects. The design details were as follows: 6locations, 2 replications/location, 2 rows/plot, row length 12 feet (+3ft alley), 9 seeds/foot, 108 seeds/row, 5 events (events 1-4 and a fifthevent that was segregating); and 4 treatments as shown below in Table 4.In all, 240 plots were planted at 6 locations (40 per location).

TABLE 4 Details of four dicamba treatments applied on transgenicsoybean. 1st Application 2nd Application Dicamba Plant Dicamba PlantTreatment Rate Stage Rate Stage 1 0 N/A 0 N/A 2 1.5 lb Preemergence 0N/A ae/Acre (Pre) 3 0 N/A 1.5 lb Postemergence ae/Acre (Post; V3-4) 41.5 lb Preemergence 1.5 lb Postemergence ae/Acre (Pre) ae/Acre (Post;V3-4)

Four non-transgenic border rows were planted all around the trial usinga known commercial line such as A3525. Optimum production and managementpractices known in the art were followed. Maximum pest control anddisease control was practiced as needed to prevent confounding effectsof dicamba applications. The field was irrigated as needed according tostandard practices.

Results shown in FIG. 5 indicate that the presence of the DMO transgeneconfers no yield penalty or other agronomic effects in soybean in theabsence of dicamba treatment. Agronomic and efficacy trials alsodemonstrated no delay in maturity of transgenic plants when comparedwith control line A3525, nor in dicamba treated plants when comparedwith untreated plant. In contrast, as shown in FIG. 6, in 4/5 events and13/15 treatments, dicamba spray showed an increase or equivalent yieldto unsprayed controls indicating a yield benefit due to improved planthealth by DCSA produced in vivo by the action of DMO on dicamba.

In the following year, transgenic soybean events comprising the DMOtransgene were field tested for yield (bu/A) in 15 locations (locs) andthe results averaged across the 15 locations are shown in FIG. 8. Thetreatments were: untreated (Untrt); 1 lb/A dicamba at planting followedby 1 lb/A dicamba at V3 growth stage (1×1); 2 lb/A dicamba at plantingfollowed by 2 lb/A dicamba at V3 growth stage (2×2); and 1 lb/A dicambaat planting followed by 1 lb/A dicamba at V3 growth stage followed by 1lb/A dicamba at R1 stage (1×1×1). Yield increases were observed at alldicamba treatments for the three events tested that ranged from 1.4 to 3bu/A or up to about 4.6 percent increase in seed yield and statisticallysignificant (5 percent confidence level, SAS 9.1) yield was measured fortreatments for events 1 and 2. In another aspect of the invention,treatment with 0.5 lb/A dicamba at planting followed by 0.5 lb/A at V3growth stage may increase yield.

Example 8 Provision of Multiple Benefits by Expressing Dicamba andGlyphosate Tolerance Genes and Treating Plants with Glyphosate andDicamba

A glyphosate tolerance gene such as CP4 EPSPS has been found to provideglyphosate tolerance and improve plant health by providing resistanceagainst several pathogens, including Phakopsora pachyrizi and Phakopsorameibomiae, the causal agents of soybean rust, which is provided bydirect action of glyphosate on such pathogenic fungi (WO05102057). Inthe present case, it was shown that DMO provides dicamba tolerance andimproves plant health by reducing pathogen and oxidative stresses.Further benefits can be obtained by combining the two genes in a singleplant by molecular and breeding methods and treating the plants withglyphosate and dicamba. This will increase the number of optionsavailable to growers depending on their market and environmental needs.

Example 9 Control of Cotton Root Knot Nematode (RKN; Meloidogyneincognita) by DCSA

DCSA was unexpectedly found to control RKN gall formation, improve plantheight and reduce number of eggs produced by the nematode in cottonplants (ST474) (Table 5). Ten to fourteen days old cotton plants weretreated with DCSA at the rate of 1 lb/A, 24 hrs before inoculating themwith RKN eggs. Cotton plants which were treated with DCSA exhibited noor low gall formation (0.6 vs 2.6), improved plant height (19.8 cm vs16.9 cm), and reduced egg count (4575 vs 6425) as compared to plantswhich were not treated with DSCA. Plant height, gall rating, and eggcounts were measured 45 days after inoculation. The gall rating scalewas 0-5 with 0=no visible galling and 5=heavy galling.

TABLE 5 Control of RKN by DCSA application in cotton. RKN alone RKN +DCSA Un-inoculated Control Plant Egg RKN Plant Egg RKN Plant RKN HeightCount/ Gall Rating Height Count/ Gall Rating Height Egg Gall RatingPlant (cm) pot (0-5) (cm) pot (0-5) (cm) Count (0-5) 1 15.9 2,160 2 25.48,160 0 21.0 0.0 0.0 2 20.3 5,120 2 25.4 9,120 0 18.4 0.0 0.0 3 12.19,480 3 25.4 5,200 0 19.7 0.0 0.0 4 20.3 5,600 3 22.2 3,720 2 20.3 0.00.0 5 15.2 2,800 3 17.8 3,360 1 14.0 0.0 0.0 6 15.2 15,400 2 14.0 3,0401 7 20.6 7,680 3 15.9 1,800 0 8 15.2 3,160 3 12.7 2,200 1 Mean 16.96,425 2.6 19.8 4,575 0.6 18.7 0.0 0.0 SE 1.1 1,557 0.2 1.9 962 0.3 1.00.0 0.0

The present invention contemplates that plant disease caused by otherplant parasitic nematodes can be reduced by treatment of plant in needof protection from nematode disease with DCSA or treatment of DMOtransgenic plants with dicamba.

Example 10 DCSA Provides Protection Against Verticillium

DCSA was unexpectedly found to provide protection to cotton plantsagainst Verticillium dahliae, causal agent of cotton wilt disease (Table6). Cotton plants of the variety ST6611, BOLLGARD 2, ROUNDUP READY FLEX,were inoculated with a moderate isolate (DPL) or a virulent isolate(King) of Verticillium by a seedling dipping method. Seedlings at thefirst true leaf stage were taken out from the pots, one inch of rootswere cut off, seedlings were dipped in a spore slurry for one minute,and then transplanted back in the pots. These strains were isolated andprovided by Dr. Terry Wheeler (Texas A&M University, TX USA). DCSA wasapplied at the rate of 1 lb/A and glyphosate (ROUNDUP WEATHERMAX,Monsanto Company, St. Louis, Mo.) at the rate of 0.75 lb/A 24 hoursbefore inoculation. Protection against Verticillium was measured on adisease severity scale of 1-9: 1 being healthy and 9 being dead becauseof disease, and also was measured as the number of plants that survivedout of 10 inoculated plants. Application of DCSA provided protectionagainst both moderate and strong isolates of Verticillium dahliae.Inoculated cotton plants that were treated with DCSA showed lower ratingand higher survival rating compared to inoculated checks that were nottreated with DCSA.

TABLE 6 Control of Verticillium by DCSA application to cotton.Verticillium (isolate DPL) Verticillium (isolate King) Plant PlantSeverity survival Severity survival rating rating rating ratingTreatment(s) (1-9) (out of 10) Treatment(s) (1-9) (out of 10) None 1 10None 1 10 Inoculated 5 8 Inoculated 9 0 check check Glyphosate 2 10Glyphosate 3.5 10 DCSA 1 10 DCSA 2 10 DCSA + 2 10 DCSA + 3 9 glyphosateglyphosate

Example 11 DCSA Provides Tolerance to Ozone in Soybeans

Many plants are sensitive to ozone (O₃), soybeans are particularlysensitive to ozone damage and show leaf flecking and chlorosis. Ozonecan be produced in the atmosphere by a photochemical process in airpolluted environments. Soybean production can be affected by ozone whensoybeans are cultivated near urban areas or air pollution from urbanareas is distributed to agricultural areas where soybeans are grown. Anexperiment was conducted to determine if DCSA treatment of soybeanplants will provide tolerance to oxidative stress caused by ozone.Soybeans were treated with 0.5 lbs/A DCSA approximately 72 hours beforeexposure to ozone. Controls had no DCSA treatment. Four plants weretreated with ozone in one 72 quart sterilite container with a clearplexiglass lid. Ozone was generated at 2 concentrations in the containerwith the plants at a low rate (2.8 ppm, parts per million) or a highrate (770 ppm) and the plants exposed for 10 hours at theseconcentrations. Light levels at plant height were ˜400 μEinsteins m-2s-1, enough to insure photosynthesis. Air in the container was mixedusing a small fan to insure proper exposure. Plants were removed fromthe treatment container and placed in a growth chamber to observesymptom development.

The results of the test were that no symptoms were observed at the lowexposure rate on the control or DCSA treated plants. Ozone visiblydamaged the lower leaves of control plants at the high ozone levelshowing typical chlorosis and leaf flecking. The DCSA treated plants didnot show any lower leaf damage at the high ozone level indicating thatDCSA protected the plants from ozone damage. In an aspect of the presentinvention, an ozone tolerance benefit is provided due to improved planthealth by DCSA applied to a plant in need of ozone tolerance or DCSAproduced in vivo by the action of DMO on dicamba in transgenic plantscomprising a DMO transgene. It is a further aspect of the invention thattreatment of

DMO containing transgenic plants with dicamba will enhance the ozonetolerance of the treated plants.

Example 12 DCSA Provides Enhanced Seed Germination

Rapid seed germination is an important agronomic trait that reduces croplosses due to seed rot especially under cool wet conditions. Soybeansseeds of three varieties were germinated in a solution of 1, 10, or 100μM DCSA or water at normal temperature (22° C.) or cool temperature (15°C.) and rated for seed germination at 1DAT (1 day after treatment) and2DAT (2 days after treatment) at 22° C. or 4DAT (days after treatment)at 15° C. The germination rate was scored as 1—low, 2—moderate, 3—highfor each treatment, 3+—exceptionally high, N=8. The results shown inTable 7 demonstrate that 1-100 μM DCSA stimulates soybean seedgermination at both normal and cool temperatures.

Wheat seed (spring wheat variety) was germinated in a solution of 0.1,1, 10, 100, or 1000 μM DCSA or water at 22° C. and rated 2 DAT forgermination using the same rating scale as used for soybean. The resultsshown in Table 8 demonstrate that wheat seed germination is enhanced inthe presence of 10-1000 μM DCSA.

In one aspect of the invention, it is contemplated that seed treatedwith DCSA or dicamba treated seed of DMO transgenic plants will haveenhanced seed germination under normal and adverse environmentalconditions.

TABLE 7 Enhanced DCSA treated soybean seed germination at 22° C. and 15°C. Treatments 22° C. and 15° C. Variety 1 Variety 2 Variety 3 Water, 1DAT, 1 1 2 22° C.  1 μM DCSA 1 2 2  10 μM DCSA 1 2 2 100 μM DCSA 3 3 1Water, 2 DAT, 2 2 2 22° C.  1 μM DCSA 3 3 2  10 μM DCSA 3 3 3 100 μMDCSA   3+   3+ 3 Water, 4 DAT, 1 1 2 15° C.  1 μM DCSA 1 3 3  10 μM DCSA3 3 2 100 μM DCSA 3 3 1

TABLE 8 Enhanced DCSA treated wheat seed germination at 22° C.Treatments, 22° C., 2 DAT Germination rating Water 1  0.1 μM DCSA 1    1μM DCSA 1   10 μM DCSA 3   100 μM DCSA 2  1000 μM DCSA 3

Example 13 DCSA Provides Enhanced Drought Tolerance

Drought tolerance is an important agronomic trait. It was found thatsoybean plants treated with DCSA showed greater tolerance to droughtconditions than the same variety not treated with DCSA. Soybean seeds oftwo varieties (1 and 2) were planted in soil in 4.5 inch pots, 3 seedsper pot. The seeds were germinated and plants grown to the V1 or V2growth stage. DCSA was prepared (0.36 grams/60 milliliters in 20 percentacetone with 10 percent surfactant) and the plants sprayed with the DCSAsolution at a rate equal to 0.5 pounds/Acre (lb/A) or 1.0 (lb/A) at day0 or day 3 of the experiment. Water was withheld at day 0. The plantswere rated on a scale of 1-4 for drought symptoms; 1—no symptoms;2—minor symptoms, leaf curling and wilting; 3—substantial symptoms andwilting; 4—dead. The treatments were surfactant only, 0.5 lb/A DCSA, and1.0 lb/A DCSA. The plants were treated with a surfactant or a DCSAsolution, water withheld, and drought symptoms rated 2 days aftertreatment (2DAT), 3 days after treatment (3DAT), and 3DAT afterrehydration (water added to soil saturation and plant recovery fromdrought symptoms measured). Fresh leaf weight (leaf grams fresh weight,leaf gfwt), fresh root weight (root grams fresh weight, root gfwt), leafdry weight (leaf grams dry weight, leaf gdwt), and root dry weight (rootgrams dry weight, root gdwt) were measured.

The average drought rating results are shown in Table 9. These resultsdemonstrate that DCSA treated plants of variety 2 showed reduced droughtsymptoms relative to the control (surfactant treated). The DCSA treatedplants of variety 2 with the reduced drought symptoms also showedgreater leaf fresh weight, greater root fresh weight and greater rootdry weight as shown in Table 10.

In one aspect of the present invention, a drought tolerance benefit isprovided due to improved plant health by DCSA applied to a plant in needof drought tolerance or DCSA produced in vivo by the action of DMO ondicamba in transgenic plants comprising a DMO transgene. It is a furtheraspect of the invention to apply dicamba to DMO containing transgenicplants to enhance drought tolerance.

TABLE 9 Enhanced drought tolerance of DCSA treated soybean plants Datacollection Treatment Variety 1 Variety 2 2 DAT surfactant 2 1.5 2 DAT0.5 lb/A DCSA 2.25 1 2 DAT 1.0 lb/A DCSA 1.75 1.25 3 DAT surfactant 3.53.5 3 DAT 0.5 lb/A DCSA 4 2.5 3 DAT 1.0 lb/A DCSA 3.75 2.5 3 DAT fbrehydration surfactant 3.25 3 3 DAT fb rehydration 0.5 lb/A DCSA 3.752.5 3 DAT fb rehydration 1.0 lb/A DCSA 3.5 2.5

TABLE 10 Enhanced leaf and root weight of DCSA treated plants underdrought stress of variety 2 Treatments Leaf gfwt Root gfwt Leaf gdwtRoot gdwt surfactant 8.01 12.88 2.37 1.16 0.5 lb/A DCSA 9.45 13.74 1.431.25 1.0 lb/A DCSA 13.94 15.40 2.20 1.34

Example 14 Enhanced Tolerance to Salt or Osmotic Stress

Transgenic soybean seeds containing the DMO gene were germinated in thepresence of 100 or 200 mM sodium chloride (NaCl, salt). The seeds hadbeen treated with 0.5 g or 2.5 g of dicamba per 100 kg of seeds. Adiglycoamine salt of dicamba (Clarity) was applied to 113.5 g samples ofsoy in a water based slurry that contained an agricultural dye. Slurryand dye rates were 8 and 0.5 fl oz/cwt. Aliquots of each dicambasolution were added to seeds contained in a glass jar and agitated.After uniform coverage was attained, seeds were placed on towels andallowed to dry. Non-DMO soybeans were treated at rates of 0, 0.5, 2.5,and 5.0 g ai/100 kg. DMO soybeans were treated with 0, 0.5 and 2.5 gai/100 kg. Seed germination was scored DAT (2 days after treatment) or5DAT (days after treatment) at 22° C. The germination rate was scored as1—low, 2—moderate, 3—high for each treatment, N=10.

The dicamba treated soybean seeds that are the progeny of DMO transgenicplants showed enhanced germination in the presence of salt relative tountreated seeds (Table 11). Additionally, a similar analysis can beconducted by treating plants with dicamba or DCSA and measuring planthealth in the presence of NaCl. Transgenic soybean plants containing theDMO gene or nontransgenic soybean plants are grown to about V1 growthstage in a growth chamber and treated with a foliar application ofsurfactant containing an equivalent of 0.5 lb/A of DCSA, 0.5 lb/A ofdicamba or a surfactant containing an equivalent of 1.0 lb/A of DCSA or1.0 lb/A of dicamba or surfactant alone. After the treatment, the plantsare subjected to osmotic stress by watering with a solution containing100 or 200 mM NaCl. The treated plants are measured for tolerance toosmotic stress by rating the stress response.

It is an aspect of the invention that seed treated with DCSA or dicambatreated seeds of DMO transgenic plants will have enhanced seedgermination under normal and adverse osmotic environmental conditionsand plants treated with DCSA or dicamba treated DMO transgenic plantswill have enhanced tolerance to salt or osmotic stress environmentalconditions. In another aspect of the invention, DCSA or dicamba treatedtransgenic seeds or plants will have a enhanced cold/freeze tolerance.

TABLE 11 Enhanced seed germination of DMO transgenic soybean seedtreated with dicamba. Treatments, 22° C., 100 mM Germination ratingGermination rating or 200 mM Nacl 2 DAT 5 DAT 100 mM NaCl, 0.0 dicamba 12 100 mM NaCl, 0.5 g ai/100 kg 2 3 dicamba 100 mM NaCl, 2.5 g ai/100 kg2 3 dicamba 200 mM NaCl, 0.0 dicamba 1 1 200 mM NaCl, 0.5 g ai/100 kg1.5 2 dicamba 200 mM NaCl, 2.5 g ai/100 kg 1.5 1 dicamba

Example 15 DCSA Provides Enhanced Tolerance to Yellow Flash in Soybeans

Yellow flash is a symptom sometimes observed in glyphosate tolerantsoybeans treated with glyphosate due to the formation of AMPA(aminomethyl phosphonic acid). A study was conducted to determine ifDCSA treatment would protect soybean plants from AMPA induced yellowflash symptoms. Soybean plants were grown to V1-2 growth stage anddivided into 3 treatment groups, 3 pots of plants per treatment. Thetreatments were 0.5 lb/A DCSA, 1.0 lb/A DCSA or surfactant alone. Oneday after treatment, all of the plants were sprayed with 1 lb/A of AMPA(Sigma Aldrich, St. Louis, Mo.) in surfactant. The plants were observedfor yellow flash symptoms eight days after treatment (8DAT) and rated ona scale of 1 (no yellow flash), 2 (moderate yellow flash) and 3 (severeyellow flash). The results, shown in Table 12, demonstrate that DCSAtreatment at 1 lb/A DCSA reduced the yellow flash symptom. In an aspectof the present invention, a tolerance to yellow flash is provided due toimproved plant health by DCSA applied to a plant in need of yellow flashtolerance or DCSA produced in vivo by the action of DMO on dicamba intransgenic plants comprising a DMO transgene.

TABLE 12 Enhanced tolerance to yellow flash in soybean. Yellow flashsymptoms Average Treatment 8 DAT, N = 3 symptoms Surfactant alone 3, 3,2 2.7 0.5 lb/A DCSA 3, 3, 3 3 1.0 lb/A DCSA 1, 2, 2 1.7

Example 16 2,4-D Tolerance Provided by DMO Transgenic Soybean

A rate titration of 2,4-D (2,4-D amine, Helena Chemical Co., Memphis,Tenn.) was applied as postemergence (POST) treatment at V3 growth stateof breeding stack of DMO soybean and RR2Y (RR2Y is MON89788 event, U.S.application Ser. No. 11/441,914) transgenic soybean. 2,4-D was appliedas a postemergence (POST) treatment (V3) at 5 application rates onDMO×RR2Y soybean stack and compared for total crop injury across allapplication rates. The POST herbicide application rates used for thistrial were 3 g ae/ha (acid equivalent/hectare), 30 g ae/ha, 60 g ae/ha,120 g ae/ha and 280 g ae/ha of 2,4-D. The treated plants were rated for2,4-D symptoms 20 days after treatment. Shown in FIG. 9, at allapplication rates, POST 2,4-D injury on the DMO×RR2Y dicamba resistantsoybean was significantly (ANOVA test) lower compared to the treatednon-transgenic control A3525. In an aspect of the present invention, aDMO transgenic soybean plant shows reduced injury from 2,4-D treatmentor accidental exposure to 2,4-D.

Example 17 Enhanced Dihaploid Production

Plant breeding is greatly facilitated by the use of doubled haploid (DH)plants. The production of DH plants enables plant breeders to obtaininbred lines without multigenerational inbreeding, thus decreasing thetime required to produce homozygous plants. A great deal of time isspared as homozygous lines are essentially instantly generated, negatingthe need for multigenerational conventional inbreeding. In particular,because DH plants are entirely homozygous, they are very amenable toquantitative genetics studies. Both additive variance and additive xadditive genetic variances can be estimated from DH populations. Otherapplications include identification of epistasis and linkage effects.Moreover, there is value in testing and evaluating homozygous lines forplant breeding programs. All of the genetic variance is among progeny ina breeding cross, which improves selection gain. Production of DHplants, which entails induction of haploidization followed bydiploidization, requires a high input of resources. DH plants rarelyoccur naturally; therefore, artificial means of production are used.First, one or more lines are generally crossed with an inducer parent toproduce haploid seed. The resulting haploid seed, which has a haploidembryo and a normal triploid endosperm, must then undergo doubling.

There are a number of well known approaches known in the art to achievechromosome doubling. Haploid cells, haploid embryos, haploid seeds,haploid seedlings, or haploid plants can be chemically treated with adoubling agent. Non-limiting examples of known doubling agents includenitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents,colchicine, pronamide, and mitotic inhibitors.

The use of these chemicals during the doubling process is toxic andstressful to the treated plant or seed. In accordance with oneembodiment of the invention, salicylic acid and its derivatives are usedto pretreatment with salicylic acid or its derivatives can enhancesurvival rate and chromosome doubling rate of seeds or plant tissuestreated with one or more of the doubling agents. To determine quantifythe effect of acetyl salicylic acid (ASA) and salicylic acid (SA) onplant health of haploid plants subjected to colchicine treatments,haploid seeds of corn genotype 01DKD2 were treated with 100-500microMolar (μM) of DCSA, ASA, or SA or combinations thereof by imbibingthe seeds in a solution of the compound for 24 hours before colchicinetreatment of the seeds. A solution of 0.0625% colchicine (Sigma Aldrich,St. Louis, Mo. Cat #C3915) and 1.5% DMSO (Sigma Aldrich, St. Louis, Mo.Cat #D8779) was used to treat the seeds and the seeds incubated in thesolution at 25° C. for 5 days in the dark. The germination rate,survival rate of the treated plants, fertility, and doubling rate weremeasured. The results shown in Table 13 demonstrate that ASA 250 μM andASA 100 μM+SA 100 μM enhanced the doubling efficiency of plantsrecovered from the colchicine treatment.

TABLE 13 ASA and SA treatment of haploid seeds imbibed for 24 hoursbefore colchicine treatment Percent 10 inch pot Percent DoublingTreatment (μM) germination survival Pollination efficiency ASA 100 87 6286 0 ASA 250 85 60 78 8 ASA 500 68 35 71 5 SA 100 80 48 83 2 SA 250 8558 85 3 SA 500 85 77 89 3 ASA 100 + SA 100 85 55 76 12 ASA 250 + SA 25082 62 70 0 ASA 500 + SA 500 85 75 84 3 Water 90 53 81 5

Alternative to seed treatment, plants at the V1-V4 growth stage can betreated with a colchicine solution containing 10-500 μM DCSA, ASA, or SAor combinations thereof as a soil drench or foliar treatment to enhancethe doubling efficiency of surviving plants.

In an aspect of the present invention, an enhanced haploid doublingefficiency is provided by treatment with salicylic acid or its analogs,which would include DCSA or DCSA produced in vivo by the action of DMOon dicamba in transgenic plants comprising a DMO transgene.

Example 18 DCSA Provides Enhanced Tolerance to Heavy Metals

Plants are sensitive to heavy metal toxicity. Heavy metals are presentin the environment or sometimes applied to plants for pest control.Symptoms of plant injury following treatment with a heavy metalcompound, for example, a compound containing copper, are necrotic leafspotting and flecking with more severe injury resulting in largerreddish brown spots, veinal necrosis and/or necrotic puckered leaves. Wetested for the ability of DCSA treatment of soybean plants to reduce thesymptoms of copper injury. Soybean seeds were planted in pots in agreenhouse and grown to the V1-2 growth stage. The plants were treatedwith DCSA and copper sulfate or copper nitrate (Fisher Scientific Co.,Pittsburg, Pa.) There were four foliar spray treatments with 4replications each: (1) 1000 ppm copper sulfate; (2) DCSA at 0.5 lb/Amixed with a 1000 ppm solution of copper sulfate applied as a tank mix;(3) DCSA at 0.5 lb/A application followed by (fb) 1 hour later treatmentof 1000 ppm solution of copper sulfate; (4) DCSA at 0.5 lb/A followed by24 hours later treatment with 1000 ppm solution of copper sulfate. Theplants were rated for percent damage 5 days after treatment.

The results shown in Table 14 demonstrate that DCSA at 0.5 lb/Asignificantly reduced 1000 ppm copper (Cu) injury on soybeans whenapplied as a tank mix (TM) or as a pretreatment at 1 hr or 24 hr beforecopper treatment. Soybean injury levels were significantly (ANOVA test)reduced from 45% to about 24-33%.

TABLE 14 Enhanced soybean tolerance to copper toxicity by DCSA treatmentTreatment Percent injury Cu 1000 ppm 45.0 a DCSA + Cu (TM) 23.8 c DCSAfb Cu (1 hr) 32.5 b DCSA fb Cu (24 hr) 32.5 b

In an aspect of the present invention, a tolerance to heavy metaltoxicity is provided due to improved plant health by DCSA applied to aplant in need of heavy metal toxicity tolerance or DCSA produced in vivoby the action of DMO on dicamba in transgenic plants comprising a DMOtransgene.

Example 19 Metabolism of Dicamba to DCSA in Soybeans

Leaf strip and whole plant studies were performed using liquidchromatography assays to determine metabolism of dicamba to DCSA insoybeans. Leaf tissue was removed from wild-type soybean plants andsoybean plants transgenic for the DMO gene and were cut into 1 mmstrips, the strips then incubated in 25 mM Tris pH 7.5 with radiolableddicamba (14C dicamba) for 24 hours at 30° C. After incubation, the leaftissue was homogenized with a spinning pestle, acidified, solidspelleted by centrifugation and an aliquot of the supernatant analyzed byRP-HPLC equipped with a radiation detector. As seen in FIG. 10, theradiolabeled dicamba was metabolized to DCSA and other polar metabolitesin the transgenic DMO gene containing soybean plants but not in thewild-type soybean plants.

For whole plant assays, soybean plants transgenic for the DMO gene weretreated with 14C dicamba by spraying the plants with a solutioncontaining the 14C dicamba at a rate up to 2.5 lb/A. Leaf samples fromthe treated plants were collected at various times after treatment andanalyzed for the DMO metabolites by RP-HPLC. The leaf tissue washomogenized with a spinning pestle, acidified, solids pelleted bycentrifugation and an aliquot of the supernatant analyze by RP-HPLCequipped with a radiation detector. As shown in FIG. 11, the whole plantassay demonstrates that in DMO gene transgenic plants DCSA is producedas a metabolite of dicamba.

Residue levels in seeds from plants treated with dicamba at planting andat V3 growth stage were very low (0.04 ppm). Residue levels increased to˜0.1 ppm with R1 application.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background, or teachmethodology, techniques, and/or compositions employed herein.

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1-32. (canceled)
 33. A method for reducing or preventing the deleterioushealth effect of a heavy metal or heavy metal salt on a plant,comprising contacting the plant with dicamba or a product ofDMO-mediated metabolism thereof in an amount that reduces or preventsthe deleterious health effect of exposure of the plant to the heavymetal or heavy metal salt.
 34. The method of claim 33, providing thedicamba or a product of DMO-mediated metabolism thereof to the plant ina composition that comprises the heavy metal or heavy metal salt. 35.The method of claim 34, wherein the composition comprises an activeingredient selected from the group consisting of a fungicide, aherbicide, a nematicide and an insecticide.
 36. The method of claim 33,further comprising the step of identifying the plant as being exposed tothe heavy metal or heavy metal salt or at risk for exposure to the heavymetal or heavy metal salt.
 37. The method of claim 33, wherein the plantis in a crop production field.
 38. The method of claim 33, wherein theheavy metal is selected from the group consisting of copper, iron,aluminum, lead, mercury, cadmium, manganese, nickel, and zinc.
 39. Themethod of claim 33, wherein the product of DMO-mediated metabolism is3,6-DCSA, 3,5-DCSA, or 3-CSA, or a metabolite of 3,6-DCSA, 3,5-DCSA, or3-CSA.
 40. The method of claim 33, wherein the product of DMO-mediatedmetabolism is an analog of DCSA.
 41. The method of claim 33, wherein theproduct is herbicidal and wherein the plant comprises a transgene thatencodes DMO.
 42. The method of claim 33, wherein the product is notherbicidal.
 43. The method of claim 33, wherein the plant is adicotyledonous plant.
 44. The method of claim 43, wherein thedicotyledonous plant is selected from the group consisting of alfalfa,beans, beet, broccoli, cabbage, carrot, cauliflower, celery, Chinesecabbage, cotton, cucumber, eggplant, flax, lettuce, lupine, melon, pea,pepper, peanut, potato, pumpkin, radish, rapeseed, spinach, soybean,squash, sugarbeet, sunflower, tomato, and watermelon.
 45. The method ofclaim 33, wherein the plant is a monocotyledonous plant.
 46. The methodof claim 45, wherein the monocotyledonous plant is selected from thegroup consisting of barley, corn, leek, onion, rice, sorghum, sweetcorn, wheat, rye, millet, sugarcane, oat, triticale, switchgrass, andturfgrass.
 47. The method of claim 33, wherein the plant is tolerant toa herbicide selected from the group consisting of glyphosate,glufosinate, 2,4-D, mesotrione, dithiopyr, isoxaflutole, bromoxynil,atrazine, fluazifop-P, and sulfonylureas/imidazolinones.
 48. The methodof claim 33, further comprising contacting the plant with at least oneherbicide selected from the group consisting of glyphosate, glufosinate,2,4-D, mesotrione, thiazopyr, isoxaflutole, bromoxynil, atrazine,fluazifop-P, and a sulfonylureas/imidazolinones. 49-55. (canceled)